Atp-based cell sorting and hyperproliferative cancer stem cells

ABSTRACT

High mitochondrial ATP is a metabolic trait that confers hyper-proliferation, sternness, anchorage-independence, anti-oxidant capacity and multi-drug resistance in cancer cells. Under the present approach, intracellular ATP levels may be used as a metabolic biomarker to identify, separate, and purify an aggressive and hyper-proliferative cancer stem cell (“CSC”) phenotype. Further, ATP may be combined with other CSC markers, e.g., CD44 or ALDH-activity, to beneficially fractionate the CSC population into sub-populations. For example, ATP-high/ CD44-high CSC sub-populations showed twice the level of anchorage-independent growth compared to ATP-low/CD44-high CSC sub-populations. Also disclosed are complementary bioinformatic data that implicate mitochondrial ATP synthesis in stemness, metastasis, and the detection of circulating tumor cells (“CTCs”), and a five-member, ATP-related metastasis gene-signature (ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB). The gene signature of the present approach may be used to identify CSCs having a dramatic increase in cell migration and invasion in vitro capacity, as well as spontaneous metastasis in vivo. The present approach also provides a cellular platform for systematically targeting sternness, multi-drug resistance, and metastasis in cancer cells.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication 62/900,139, filed Sep. 13, 2019, and incorporated herein byreference in its entirety.

FIELD

The present disclosure relates to ATP-based cell sorting to identify,separate, and treat metabolically-hyperactive, aggressive, andhyper-proliferative cancer stem cell (“CSC”) phenotypes, and forpreventing or reducing the likelihood of metastasis.

BACKGROUND

Researchers have struggled to develop new anti-cancer treatments.Conventional cancer therapies (e.g. irradiation, alkylating agents suchas cyclophosphamide, and anti-metabolites such as 5-Fluorouracil) haveattempted to selectively detect and eradicate fast-growing cancer cellsby interfering with cellular mechanisms involved in cell growth and DNAreplication. Other cancer therapies have used immunotherapies thatselectively bind mutant tumor antigens on fast-growing cancer cells(e.g., monoclonal antibodies). Unfortunately, tumors often recurfollowing these therapies at the same or different site(s), indicatingthat not all cancer cells have been eradicated. Relapse may be due toinsufficient chemotherapeutic dosage and/or emergence of cancer clonesresistant to therapy. Hence, novel cancer treatment strategies areneeded.

Advances in mutational analysis have allowed in-depth study of thegenetic mutations that occur during cancer development. Despite havingknowledge of the genomic landscape, modern oncology has had difficultywith identifying primary driver mutations across cancer subtypes. Theharsh reality appears to be that each patient's tumor is unique, and asingle tumor may contain multiple divergent clone cells. What is needed,then, is a new approach that emphasizes commonalities between differentcancer types. Targeting the metabolic differences between tumor andnormal cells holds promise as a novel cancer treatment strategy. Ananalysis of transcriptional profiling data from human breast cancersamples revealed more than 95 elevated mRNA transcripts associated withmitochondrial biogenesis and/or mitochondrial translation. Additionally,more than 35 of the 95 upregulated mRNAs encode mitochondrial ribosomalproteins (MRPs). Proteomic analysis of human breast cancer stem cellslikewise revealed the significant overexpression of severalmitoribosomal proteins as well as other proteins associated withmitochondrial biogenesis.

Mitochondria are extremely dynamic organelles in constant division,elongation and connection to each other to form tubular networks orfragmented granules in order to satisfy the requirements of the cell andadapt to the cellular microenvironment. The balance of mitochondrialfusion and fission dictates the morphology, abundance, function andspatial distribution of mitochondria, therefore influencing a plethoraof mitochondrial-dependent vital biological processes such as ATPproduction, mitophagy, apoptosis, and calcium homeostasis. In turn,mitochondrial dynamics can be regulated by mitochondrial metabolism,respiration and oxidative stress. Thus, it is not surprising that animbalance of fission and fusion activities has a negative impact onseveral pathological conditions, including cancer. Cancer cells oftenexhibit fragmented mitochondria, and enhanced fission or reduced fusionis often associated with cancer, although a comprehensive mechanisticunderstanding on how mitochondrial dynamics affects tumorigenesis isstill needed.

An intact and enhanced metabolic function is necessary to support theelevated bioenergetic and biosynthetic demands of cancer cells,particularly as they move toward tumor growth and metastaticdissemination. Not surprisingly, mitochondria-dependent metabolicpathways provide an essential biochemical platform for cancer cells, byextracting energy from several fuels sources.

Cancer stem-like cells are a relatively small sub-population of tumorcells that share characteristic features with normal adult stem cellsand embryonic stem cells. As such, CSCs are thought to be a ‘primarybiological cause’ for tumor regeneration and systemic organismal spread,resulting in the clinical features of tumor recurrence and distantmetastasis, ultimately driving treatment failure and premature death incancer patients undergoing chemo- and radio-therapy. Evidence indicatesthat CSCs also function in tumor initiation, as isolated CSCsexperimentally behave as tumor-initiating cells (TICs) in pre-clinicalanimal models. As approximately 90% of all cancer patients diepre-maturely from metastatic disease world-wide, there is a greaturgency and unmet clinical need, to develop novel therapies foreffectively targeting and eradicating CSCs. Most conventional therapiesdo not target CSCs and often increase the frequency of CSCs, in theprimary tumor and at distant sites.

Recently, energetic metabolism and mitochondrial function have beenlinked to certain dynamics involved in the maintenance and propagationof CSCs, which are a distinguished cell sub-population within the tumormass involved in tumor initiation, metastatic spread and resistance toanti-cancer therapies. For instance, CSCs show a peculiar and uniqueincrease in mitochondrial mass, as well as enhanced mitochondrialbiogenesis and higher activation of mitochondrial protein translation.These behaviors suggest a strict reliance on mitochondrial function.Consistent with these observations, an elevated mitochondrial metabolicfunction and OXPHOS have been detected in CSCs across multiple tumortypes.

One emerging strategy for eliminating CSCs exploits cellular metabolism.CSCs are among the most energetic cancer cells. Under this approach, ametabolic inhibitor is used to induce ATP depletion and starve CSCs todeath. So far, the inventors have identified numerous FDA-approved drugswith off-target mitochondrial side effects that have anti-CSC propertiesand induce ATP depletion, including, for example, the antibioticDoxycycline, which functions as a mitochondrial protein translationinhibitor. Doxycycline, a long-acting Tetracycline analogue, iscurrently used for treating diverse forms of infections, such as acne,acne rosacea, and malaria prevention, among others. In a recent Phase IIclinical study, pre-operative oral Doxycycline (200 mg/day for 14 days)reduced the CSC burden in early breast cancer patients between 17.65%and 66.67%, with a near 90% positive response rate.

However, certain limitations restrain the use of sole anti-mitochondriaagents in cancer therapy, as adaptive mechanisms can be adopted in thetumor mass to overcome the lack of mitochondrial function. Theseadaptive mechanisms include, for example, the ability of CSCs to shiftfrom oxidative metabolism to alternate energetic pathways, in amulti-directional process of metabolic plasticity driven by bothintrinsic and extrinsic factors within the tumor cells, as well as inthe surrounding niche. Notably, in CSCs the manipulation of suchmetabolic flexibility can turn as advantageous in a therapeuticperspective. What is needed, then are therapeutic approaches that eitherprevent these metabolic shifts, or otherwise take advantage of the shiftto inhibit cancer cell proliferation.

Adenosine-5′-triphosphate (ATP) is the bio-energetic “currency” of allliving cells and organisms. Chemically, ATP is a nucleosidetriphosphate, which contains adenine, a ribose sugar, and threephosphate groups. ATP cleavage at its terminal phosphate group, producestwo main reaction products, ADP and inorganic phosphate (Pi), therebyreleasing high levels of stored energy. In eukaryotic cells,mitochondria generate the vast amount of ATP via the TCA cycle andoxidative phosphorylation (OXPHOS), while glycolysis contributes a minoramount of ATP. Mitochondrial dysfunction induces ATP-depletion,resulting in mitochondrial-driven apoptosis (cell death).

In MCF7 breast cancer cells, mitochondrial-driven OXPHOS contributes to80% of ATP production, while glycolysis contributes the remaining 20%.Therefore, like normal cells, cancer cells are still highly dependent onmitochondrial ATP production. However, it remains largely unknown howATP levels in cancer cells contribute to “stemness” and cell cycleprogression, as well as their ability to undergo anchorage-independentgrowth, a characteristic feature of metastatic spread.

Because of the central importance of ATP as a barometer of cellmetabolism, many luminescent and fluorescent probes have been developedto measure and track ATP levels, in response to various cellularstimuli. For example, ATP-Red 1 (CAS#: 1847485-97-5, IUPAC Name: [2-[3′,6′-bis(diethylamino)-3-oxospiro[isoindole-1,9′-xanthene]-2-yl]phenyl]boronicacid) is a vital dye that is only fluorescent when bound to ATP, anddoes not recognize ADP or other nutrients. ATP-Red 1 allows for thedynamic visualization of ATP levels in living cells and tissues.

An object of this disclosure is to describe a viable ATP-depletionstrategy for targeting and eradicating even the “fittest” cancer cells.

It is another object of this disclosure to describe unique compositionsof cells of a particular, hyper-proliferative phenotype.

It is another object of this disclosure to identify new anti-cancertherapeutic approaches involving new pharmaceutical compounds thatmetabolically starve CSCs by targeting mitochondria and driving ATPdepletion.

SUMMARY

The present approach describes the use of a fluorescent ATP imagingprobe to metabolically fractionate a cancer cell population, andseparate a hyper-proliferative cell sub-population. The resultingcomposition may be used for numerous advantageous purposes, ranging fromrapid drug development and screening, to predicting and preventingmetastasis and drug resistance. The present approach also provides a5-gene signature prognostic of metastasis in a cancer, and methods formetabolic fractionation of cancer cells, and diagnosis and prevention ofmetastasis.

Bioenergetic cell “stratification” employing an ATP-based biomarker maybe used to isolate the “fittest” cancer cells, for identification,diagnosis, treatment, and therapeutic drug development. In particular, afluorescent ATP imaging probe, such as Biotracker ATP-Red 1, may be usedto stain a cell population, and the resulting ATP-based fluorescence maybe used to metabolically fractionate the population into ATP-high and,if desired, bulk and ATP-low sub-populations. Using this novel approach,the data disclosed herein includes the first evidence that high levelsof mitochondrial ATP are a primary determinant of aggressive cancer cellbehavior(s), including spontaneous metastasis.

There is a considerable amount of phenotypic diversity and metabolicheterogeneity in the cancer cell population. This heterogeneity allowsthe “fittest” cancer cells to escape current treatment modalities,resulting in tumor recurrence and distant metastasis, secondary to drugresistance.

High intracellular ATP levels may be used as a metabolic biomarker foran aggressive and hyper-proliferative cancer cell phenotype. Under thepresent approach, a fluorescent ATP marker, such as the vital dyeBioTracker™ ATP-Red 1 (EMD Millipore Corporation, Burlington,Massachusetts), may be used to quantify mitochondrial ATP levels in acancer cell population, and isolate ATP-high and ATP-low cancer cellsub-populations by flow cytometry. Phenotypic analysis of thesesub-populations shows that high mitochondrial ATP is a metabolic traitthat confers hyper-proliferation, sternness, anchorage-independence,anti-oxidant capacity, and multi-drug resistance in cancer cells.Quantitatively similar results were obtained with four human breastcancer cell lines, MCF7, T47D, MDA-MB-231 and MDA-MB-468.

By combining ATP with other CSC markers, e.g., CD44 or ALDH-activity,the CSC population may be advantageously fractionated into twosub-populations. The CD44-high/ATP-high sub-populations have about twicethe level of anchorage-independent growth compared to CD44-high/ATP-lowsub-populations. Thus, CD44-high/ATP-low cancer cells represent a moredormant CSC population. Importantly, these results indicate that ATPlevels may be a functional regulator of dormancy in CSCs.

The present approach also includes complementary bioinformatic data thatimplicate mitochondrial ATP synthesis in stemness, metastasis, and thedetection of circulating tumor cells (CTCs). Disclosed herein is afive-member, ATP-related metastasis gene-signature comprising ABCA2,ATP5F1C, COX20, NDUFA2 and UQCRB. In accordance with thesemetastasis-based clinical findings, ATP-high MDA-MB-231 cells showeddramatic increases in their capacity to undergo both cell migration andinvasion in vitro, as well as spontaneous metastasis in vivo.

Thus, the present approach provides a new cellular platform forsystematically identifying, studying, and targeting stemness, multi-drugresistance, and metastasis in cancer cells. This disclosure alsomechanistically explains the positive therapeutic benefits of i)nutrient fasting and ii) caloric-restriction mimetics, for improvingcancer therapy, by inducing ATP-depletion.

In embodiments of the present approach, vital dye ATP-Red 1 is used as amolecular probe to identify and isolate ATP-high and ATP-lowsub-populations of cells, and more specifically, cancer cells and CSCs.The ATP-high sub-population of cancer cells are larger, more energetic,hyper-proliferative and undergo anchorage-independent growth, consistentwith a more “stem-like” phenotype. These ATP-rich cells may be targetedwith ATP-depletion therapy, to eradicate the energetically “fittest”CSCs, reduce drug resistance, and prevent metastasis.

Some embodiments of the present approach may take the form of a purifiedcomposition of hyper-proliferative cancer stem cells, in the form of asub-population of cells from a human cancer cell population, the cancercell population expressing a range of fluorescent signals in response toa fluorescent adenosine triphosphate (ATP) imaging probe, and thesub-population of cells expressing an upper portion of the range ofATP-based fluorescent signals. The fluorescent ATP imaging probe may be,for example, BioTracker ATP-Red 1. The upper portion, or ATP-highsub-population, may be the top 10%, 5%, or 1% of ATP-based fluorescentsignals, depending on the embodiment. Other portions may be used. Insome embodiments, the composition is positive for a CD44 marker. In someembodiments, the composition is positive for an ALDH marker. In someembodiments, the composition is frozen.

In some embodiments, the present approach may take the form of apurified cell composition comprising a cancer stem cell sub-populationstained with a fluorescent ATP imaging probe and expressing a targetportion of an ATP-based fluorescent signal range of a cancer cellpopulation. The cancer cell population expresses a range of ATP-basedfluorescent signals, and the target portion of the ATP-based fluorescentsignal range may be an upper portion of the ATP-based fluorescentsignals (e.g., ATP-high sub-population) and/or a lower portion of theATP-based fluorescent signals (e.g., ATP-low sub-population). The targetportion may be the top or bottom 10%, 5%, or 1% of ATP-based fluorescentsignals, or other portion as selected.

Some embodiments may take the form of a purified composition of cellsobtained by staining a human cancer cell population with a fluorescentATP imaging probe, separating a fraction of the human cancer cellpopulation having a target portion of ATP-based fluorescent signals, andpurifying the separated cells. The target portion may be, for example,the top 10% of ATP-based fluorescent signals, the top 5% of ATP-basedfluorescent signals, the bottom 10% of ATP-based fluorescent signals,the bottom 5% of ATP-based fluorescent signals, etc. The separated cellsare positive for one of a CD44 marker and an ALDH marker.

Some embodiments may take the form of a method of ATP-based cellfractionation. Cells in a cell population may be stained with afluorescent ATP imaging probe that fluoresces when bound to ATP. TheATP-based fluorescent signals of the stained cells in the cellpopulation may be measured. The stained cells may be separated based ona target portion of ATP-based fluorescent signals.Fluorescence-activated cell sorting (FACS) and gating of the targetportion of ATP-based fluorescent signals may be used to separate thestained cells. The gates may be set to collect the stained cells havingthe top 10% of measured fluorescent signals, and/or the stained cellshaving the bottom 10% of measured fluorescent signals. It should beappreciated that other percentages may be used. The cell population maybe derived from, for example, of blood, urine, saliva, tumor tissue,non-cancerous tissue, or a metastatic lesion. Some embodiments mayfurther include measuring ALDH activity of separated cells, measuringanchorage-independent growth of separated cells, measuring themitochondrial mass of separated cells, measuring the glycolytic andoxidative mitochondrial metabolism of separated cells, measuring thecell cycle progression and proliferative rate of separated cells, andmeasuring the poly-ploidy of separated cells.

Embodiments of the present approach may take the form of a method forseparating and collecting metabolically-active cells from a cellpopulation. Cells in a cell population may be stained with anATP-labeling dye that fluoresces when bound to ATP. The fluorescentsignals of the stained cells may be measured in the cell population, andthen the stained cells based on the measured fluorescent signals. Atleast a portion of the separated cells, having a measured fluorescentsignal one of above a predetermined threshold and below a predeterminedthreshold, may then be collected, such as by using a FACS machine. Thepredetermined threshold comprises a percentage of an upper portion ofthe measured fluorescent signals, such as, for example, the top 25%, thetop 20%, the top 15%, the top 10%, the top 5%, the top 2%, and the top1%. Other percentages may be used without departing from the presentapproach. In some embodiments, the separated cells may be furtherseparated based on a second marker, such as CD44(+), CD133(+), ESA(+),ALDEFLOUR(+), MitoTracker-High, EpCAM(+), CD90(+), CD34(+), CD29(+),CD73(+), CD90(+), CD105(+), CD106(+), CD166(+), and Stro-1(+). Othermarkers may be used, without departing from the present approach. Thesecond marker may take the form of an antibody coated on magnetic beads,in some embodiments.

The present approach may also take the form of a method for identifyingand treating cancer stem cells in a biologic sample. A biologic samplemay be obtained from a patient, and then cells in the biologic samplemay be stained with an ATP-labeling dye, wherein the ATP-labeling dyefluoresces when bound to ATP. The fluorescent signals of the stainedcells in the cell population may be measured, and then compared to apredetermined threshold indicating the presence of cancer stem cells. Ifthe measured fluorescent signals exceeds the predetermined threshold, anATP-depletion therapeutic may be administered to the patient. TheATP-depletion therapeutic may be, for example, Doxycycline, Tigecycline,Azithromycin, Pyrvinium pamoate, Atovaquone, Bedaquiline, Niclosamide,Irinotecan, Actinonin, CAPE, Berberine, Brutieridin, Melitidin,Oligomycin, AR-C155858, a Mitoriboscin, a Mitoketoscin, a Mitoflavoscin,a TPP-derivative, dodecyl-TPP, 2-Butene-1,4-bis-TPP, or the combinationof Doxycycline, Azithromycin and Ascorbic acid.

In some embodiments, the present approach may take the form of a methodof testing a candidate compound for anti-cancer activity. A cancer cellpopulation may be stained with an ATP-labeling dye that fluoresces whenbound to ATP, such as BioTracker ATP-Red 1. The ATP-based fluorescentsignals of the stained cells may be measured, and the stained cells maybe separated based on a target portion of ATP-based fluorescent signalsto prepare a hyper-active cancer cell sub-population. The candidatecompound may be administered to the hyper-active cancer cellsub-population; the effect of the candidate compound on the hyper-activecancer cell sub-population may be measured. The ATP-labeling dye may beBioTracker ATP-Red 1. The target portion of ATP-based fluorescentsignals may be, for example, the top 25%, the top 20%, the top 15%, thetop 10%, the top 5%, the top 2%, and the top 1%. In some embodiments,the hyper-active cancer cell sub-population is positive for one of aCD44 marker an ALDH marker. Embodiments may also involve measuring ALDHactivity of the hyper-active cancer cell sub-population, measuringanchorage-independent growth of the hyper-active cancer cellsub-population cells, measuring the mitochondrial mass of thehyper-active cancer cell sub-population, measuring the glycolytic andoxidative mitochondrial metabolism of the hyper-active cancer cellsub-population, measuring the cell cycle progression and proliferativerate of the hyper-active cancer cell sub-population, and measuring thepoly-ploidy of the hyper-active cancer cell sub-population.

The present approach may also take the form of a method of diagnosingand preventing a risk of metastasis in a cancer patient. The expressionlevels of the 5-member gene signature of ABCA2, ATP5F1C, COX20, NDUFA2,and UQCRB, in a biologic sample of the patient's cancer may bedetermined, and then compared to baseline expression levels of ABCA2,ATP5F1C, COX20, NDUFA2, and UQCRB, in a non-cancerous biologic samplefrom the patient. If the detected expression levels exceed the baselineexpression levels, an ATP-depletion compound may be administered to thepatient. The ATP-depletion compound may be, for example, Doxycycline,Tigecycline, Azithromycin, Pyrvinium pamoate, Atovaquone, Bedaquiline,Niclosamide, Irinotecan, Actinonin, CAPE, Berberine, Brutieridin,Melitidin, Oligomycin, AR-C155858, a Mitoriboscin, a Mitoketoscin, aMitoflavoscin, a TPP-derivative, dodecyl-TPP, 2-Butene-1,4-bis-TPP, or acombination of Doxycycline, Azithromycin and Ascorbic acid.

Some embodiments may take the form of a kit for identifying circulatingtumor cells in a biologic sample. The kit may include reagents foridentifying an up-regulation of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRBin the biologic sample, such as antibodies directed to the proteinsencoding those genes. The kit may be used for, as an example, a liquidbiopsy procedure to detect CTCs.

The present approach may also take the form of a method for detectingcirculating tumor cells (CTCs) in a biologic sample. The expressionlevels of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB, in the biologicsample may be determined, and then CTCs are identified as present if thedetermined expression levels are upregulated relative to a control. Thebiologic sample may be, as examples, blood, urine, saliva, tumor tissue,non-cancerous tissue, or a metastatic lesion. The sample may be furtherprocessed to separate ATP-high cells, using the methods describedherein.

These and other embodiments will be apparent to the person having anordinary level of skill in the art in view of this description, theclaims appended hereto, and the applications incorporated by referenceherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a HeatMap of ATP-related genes that were transcriptionallyupregulated under both 3D growth conditions (anchorage-independent andin vivo tumors), all relative to 2D-adherent growth. FIGS. 1B and 1Cshow volcano plots for the GSE2034 and GSE59000 GEO DataSets. FIG. 1Dshows a Venn diagram intersecting the two breast cancer metastasis GEODataSets (GSE2034 and GSE59000), used to identify ATP-related geneshighly upregulated in both data sets, as prognostic biomarkers ofmetastasis.

FIGS. 2A-2N are data plots showing the positive correlation of APT5F1Cversus the genes CDH1, ALDH2, SOX2, VIM, CD44, EPCAM, MKI67, RRP1B,CXCR4, VCAM1, CDK1, CDK2, CDK4, and CDK6, respectively. FIGS. 20-2Q aredata plots showing the positive correlation of APT5F1C versus UQCRB,COX20, and NDUFA2, respectively.

FIG. 3A shows a Kaplan-Meier curve for ER(+), recurrence-free survival(N=3,082), FIG. 3B shows a Kaplan-Meier curve for ER(+), distantmetastases-free survival (N=1,395), and FIG. 3C shows a Kaplan-Meiercurve for ER(+), lymph node negative, Tamoxifen-treated, relapse-freesurvival (N=471).

FIG. 4A shows a HeatMap of an ATP-ABC gene expression profile, and FIG.4B shows a HeatMap of the OXPHOS gene expression profile. FIG. 4C is aWestern blot analysis of MDA-MB-231 cells in the ATP-high and ATP-lowsub-populations.

FIG. 5A illustrates an embodiment of the metabolic fractionationprocedure according to the present embodiment. FIG. 5B illustrates anexample of metabolic fractionation of MCF7 cells with ATP-Red 1, toisolate ATP-high (top 5%) and ATP-low (bottom 5%) cell sub-populations.

FIGS. 6A and 6B show results from a continuous, real-time assay systemon cell proliferation of three cell sub-populations (ATP-low 5%, Bulk5%, ATP-high 5%).

FIG. 7A is a bar graph that shows changes in luminescence of cells inthe ATP-high MCF7 sub-population, and FIG. 7B shows mammosphereformation assay results for ATP-high, bulk, and ATP-low sub-populations.FIG. 7C shows comparative images of the cell sub-populations after theassay. FIG. 7D shows signal strength for CD44 and ALDH positivesub-populations, and FIG. 7E shows the results of the Cell-Titer-Glo ofthis analysis.

FIGS. 8A and 8B show results relating to the metabolic profiling of3D-mammospheres and ATP-high MCF7 cells.

FIGS. 9A and 9B show Cell-Titer-Glo and 3D mammosphere formation resultsfor ATP-high and ATP-low sub-populations of MCF7, T47D, MDA-MB-231 andMDA-MB-468 cells, using a 10% gate.

FIG. 10A shows luminescence in ATP-high and ATP-low sub-populations(10%) in a MCF7 cell population after a 24-hour period. FIGS. 10Bthrough 10E show the results of metabolic flux analysis on the ATP-highand ATP-low sub-populations.

FIGS. 11A-11D show cell cycle progression in MCF7, T47D, MDA-MB-468, andMDA-MB-231 cells, using FACS analysis with propidium iodide to detectDNA-content.

FIG. 12A shows drug resistance results for the ATP-low sub-population(bottom 5%), and FIG. 12B shows drug resistance results for the ATP-highsubpopulation (top 5%).

FIGS. 13A and 13B show mammosphere assay formation results fordouble-labelled cells (CD44 and ATP) in MCF7 cells and MDA-MB-231 cells,respectively, and FIGS. 13C and 13D show mammosphere assay formationresults for double-labelled cells (ALDH-activity and ATP) in in MCF7cells and MDA-MB-231 cells, respectively.

FIGS. 14A and 14B show the results of a migration and invasion assay onMDA-MB-231 cells in an ATP-high sub-population.

FIG. 15 shows the results of the spontaneous metastasis in vivo CAMassay.

FIGS. 16A-16C show luminescence change, cell cycle progression, andmammosphere formation assay results of Tempo-ATP MCF7 cells,respectively.

DESCRIPTION

The following description illustrates embodiments of the presentapproach in sufficient detail to enable practice of the presentapproach. Although the present approach is described with reference tothese specific embodiments, it should be appreciated that the presentapproach can be embodied in different forms, and this description shouldnot be construed as limiting any appended claims to the specificembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the present approach to those skilled in the art.

This description uses various terms that should be understood by thoseof an ordinary level of skill in the art. The following clarificationsare made for the avoidance of doubt. The terms “treat,” “treated,”“treating,” and “treatment” include the diminishment or alleviation ofat least one symptom associated or caused by the state, disorder ordisease being treated, in particular, cancer. In certain embodiments,the treatment comprises diminishing and/or alleviating at least onesymptom associated with or caused by the cancer being treated, by thecompound of the invention. In some embodiments, the treatment comprisescausing the death of a category of cells, such as CSCs, of a particularcancer in a host, and may be accomplished through preventing cancercells from further propagation, and/or inhibiting CSC function through,for example, depriving such cells of mechanisms for generating energy.For example, treatment can be diminishment of one or several symptoms ofa cancer, or complete eradication of a cancer. As another example, thepresent approach may be used to inhibit mitochondrial metabolism in thecancer, eradicate (e.g., killing at a rate higher than a rate ofpropagation) CSCs in the cancer, eradicate TICs in the cancer, eradicatecirculating tumor cells in the cancer, inhibit propagation of thecancer, target and inhibit CSCs, target and inhibit TICs, target andinhibit circulating tumor cells, prevent (i.e., reduce the likelihoodof) metastasis, prevent recurrence, sensitize the cancer to achemotherapeutic, sensitize the cancer to radiotherapy, sensitize thecancer to phototherapy.

The terms “cancer stem cell” and “CSC” refer to the subpopulation ofcancer cells within tumors that have capabilities of self-renewal,differentiation, and tumorigenicity when transplanted into an animalhost. Compared to “bulk” cancer cells, CSCs have increased mitochondrialmass, enhanced mitochondrial biogenesis, and higher activation ofmitochondrial protein translation. As used herein, a “circulating tumorcell” is a cancer cell that has shed into the vasculature or lymphaticsfrom a primary tumor and is carried around the body in the bloodcirculation. The CellSearch Circulating Tumor Cell Test may be used todetect circulating tumor cells.

The phrases “ATP-high” and “ATP-low” refer to cell sub-populationshaving ATP-based fluorescent signals representing the upper and lowerportions of the ATP-based fluorescent signals, respectively, from astarting cell population. The upper portion may represent the top 25% ofthe starting cell population's ATP-based fluorescent signals, or the top20%, or the top 15%, or the top 10%, or the top 5%, or the top 2%, orthe top 1%. The lower portion may represent the bottom 25% of thestarting cell population's ATP-based fluorescent signals, or the bottom20%, or the bottom 15%, or the bottom 10%, or the bottom 5%, or thebottom 2%, or the bottom 1%.

The phrase “pharmaceutically effective amount,” as used herein,indicates an amount necessary to administer to a host, or to a cell,tissue, or organ of a host, to achieve a therapeutic result, such asregulating, modulating, or inhibiting protein kinase activity, e.g.,inhibition of the activity of a protein kinase, or treatment of cancer.A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

Bioinformatics analysis demonstrates the role of mitochondrial ATPsynthesis, in 3D anchorage-independent growth, stemness, and distantmetastasis. In particular, mitochondrial ATP synthesis is a keydeterminant of 3D anchorage-independent growth and metastasis, using abioinformatics approach. Existing proteomic profiling data wasinterrogated to compare 2D-monolayers with 3D-mammospheres, in twodistinct ER(+) breast cancer cell lines (MCF7 and T47D). Overall, from1,519 common proteins in both cell lines, 21 ATP-related proteins werefound to be up-regulated in both data sets, in 3D-mammospheres. Table 1,below, shows these proteins, with accession number, and the fold changein expression in MCF7 and T47D cells (spheres versus 2-D adherentgrowth). Out of these 21 ATP-related proteins, 7 subunits of themitochondrial ATP-synthase were detected, including ATP5F1B, ATP5F1C,ATP5IF1, ATPSMG, ATPSPB, ATPSPD and ATPSPO. Using Ingenuity PathwayAnalysis (IPA) Software, we observed that the predicted upstreamregulators of 3D anchorage-independent growth were highly conservedbetween the two cell lines and were specifically associated withexisting IPA data sets, related to tumor growth and tumor cellproliferation.

TABLE 1 The 21 ATP-related proteins up-regulated in 3D- mammospheres forboth MCF7 and T47D cell lines. Expr Fold Change MCF7 Expr Fold ChangeT47D 3D Spheres v. 3D Spheres v. Symbol Accession 2D Adh. 2D Adh. ABCF3B4DRU9 358.862 3.849 ATP13A2 Q8NBS1 560.751 42.918 ATP13A4 H7C1P5 20.51725.583 ATP1A1 B7Z3V1 51.419 14.905 ATP1A3 P13637 229.056 16.789 ATP1A4Q13733 440.915 60.871 ATP1B1 A3KLL5 1.913 10.585 ATP1B3 P54709 12.0823.433 ATP2A2 P16615 20.836 7.006 ATP2A3 Q93084 78.477 23.288 ATP2B1E7ERY9 7.655 2.928 ATP5F1B Q0QEN7 10.129 2.087 ATP5F1C Q8TAS0 1.9471.634 ATP5IF1 Q9UII2 10.127 16.359 ATP5MG O75964 1.619 1.429 ATP5PBQ53GB3 2.515 4.379 ATP5PD O75947 2.27 1.436 ATP5PO P48047 1.92 1.426COX5B P10606 7.692 1.513 NDUFAB1 H3BNK3 87.149 11.95 UQCR10 Q9UDW1 2.4571.623 UQCRH Q567R0 1.662 2.171

We also re-analyzed GEO transcriptional profiling data sets, comparing2D-growth, 3D-growth, and the in vivo tumor growth of MDA-MB-231 cells(a triple-negative breast cancer cell line). FIG. 1A shows a HeatMap ofATP-related genes that were transcriptionally upregulated under both 3Dgrowth conditions (anchorage-independent and in vivo tumors), allrelative to 2D-adherent growth. The first column identifies the gene,the second column shows the expression profile in 2D MDA-MB-231 cells,the third column shows the expression profile in 3D MDA-MB-231 cells,and the fourth column shows the expression profile in xenograftMDA-MB-231 cells. Darker cells indicate less fold change, and lightercells indicate higher fold change. The HeatMap shows the log of the foldchange, e.g., the lightest cells are +/−4. In the 2D MDA-MB-231 column,lighter cells indicate a negative change (e.g., ATP11A-AS1 showed a -4log fold change), whereas lighter cells in the 3D and xenograft columnsindicate a positive change (e.g., ATP12A showed a 4 log fold change).

The transcriptional expression of ATP-related genes (OXPHOS andATP-related transporters) in two distinct GEO DataSets related to humanbreast cancer metastasis were useful for identifying ATP-related genesassociated with metastasis. FIGS. 1B and 1C show volcano plots for theGSE2034 and GSE59000 GEO DataSets. Specifically, FIG. 1B compares geneexpression in scenarios with metastasis versus scenarios with nometastasis (GSE2034), and FIG. 1C compares gene expression in scenarioswith metastasis versus the primary tumor (GSE59000). The volcano plotswere produced by examining the annotations present in OncoLandMetastatic Cancer (QIAGEN OmicSoft Suite) and by performing functional“core analyses” using Ingenuity Pathway Analysis Software (IPA; QIAGEN),on genes annotated with an uncorrected p-value cut off <0.05. Thetranscriptional profiles of ATP-related genes (OXPHOS and ATP-relatedtransporters), were increased and specifically associated withmetastasis, in both GEO DataSets.

FIG. 1D shows a Venn diagram intersecting the two breast cancermetastasis GEO DataSets (GSE2034 and GSE59000), used to identifyATP-related genes highly upregulated in both data sets, as prognosticbiomarkers of metastasis. The intersection of the two GEO DataSets wasperformed, as described in connection with FIGS. 1B and 1C, using IPASoftware. The overlapping set of 1,055 genes contained only 5ATP-related genes. These ATP-related genes, ABCA2, ATP5F1C, COX20,NDUFA2, and UQCRB, were highly upregulated in both metastasis GEODataSets, and thus have prognostic value with respect to predictingmetastasis of a cancer. These five ATP-related genes may be used as anATP-related metastasis gene-signature, prognostic of metastasis. Mostnotably, ATP5F1C (also known as ATP5C1) encodes the gamma-subunit of thesoluble Fl-catalytic core of the mitochondrial ATP synthase. UQCRB isthe essential component of mitochondrial complex III, which functionallybinds ubiquinone and participates in electron transport. COX20 is achaperone that is essential for the assembly of mitochondrial complexIV. NDUFA2 is essential for the function of mitochondrial complex I.Finally, ABCA2 is a member of the ATP-binding cassette transporter genefamily.

In bona fide breast cancer metastatic lesions, ATP5F1C transcriptionalexpression is positively correlated with the co-expression of: i) fivemetastatic marker genes (EPCAM, MKI67, RRP1B, VCAM1, CXCR4); ii) fourcell cycle regulatory genes (CDK1, CDK2, CDK4, CDK6); and iii) elevenCSC marker genes (CDH1, ALDH2, ALDH1BA1, ALDH9A1, SOX2, VIM, CDH2,ALDH7A1, ALDH1B1, CD44, ALDH3B2, listed in rank order of statisticalsignificance). FIGS. 2A-2N are data plots showing the positivecorrelation of APT5F1C versus each of these genes, in the order of CDH1,ALDH2, SOX2, VIM, CD44, EPCAM, MKI67, RRP1B, CXCR4, VCAM1, CDK1, CDK2,CDK4, and CDK6.

Additionally, ATP5F1C transcriptional expression is also positivelycorrelated with the co-expression of mitochondrial complexes I-V, mt-DNAencoded transcripts and three other members of the five-membermetastasis gene-signature, namely UQCRB, COX20 and NDUFA2. FIGS. 20-2Qare data plots showing the positive correlation of APT5F1C versus UQCRB,COX20, and NDUFA2, respectively. The expression of two members of thismetastasis gene signature, ATP5F1C and UQCRB, has been functionallycorrelated with maximal oxygen uptake (Vo2max) and a high percentage oftype 1 fibers (mitochondrial-rich) in human skeletal muscle tissues.

The expression of ATP5F1C in skeletal muscle is also increasedsignificantly after exercise training, reflecting increased musclefitness in patients. Conversely, ATP5F1C levels decreased with advancedage and were reduced in progeria syndrome patients. These results arehighly suggestive that high ATP5F1C expression is a biomarker ofincreased mitochondrial ATP production at the cellular level.

Using Kaplan-Meier (K-M) analysis, we determined that ATP5F1C is aprognostic biomarker for distant metastasis and tumor recurrence,especially in ER(+) patients that are lymph node negative at diagnosisand were treated with Tamoxifen (Hazard Ratio (recurrence-freesurvival)=2.77; P=3.4E-06; N=471). FIG. 3A shows the Kaplan-Meier curvefor ER(+), recurrence-free survival (N=3,082), FIG. 3B shows theKaplan-Meier curve for ER(+), distant metastases-free survival(N=1,395), and FIG. 3C shows the Kaplan-Meier curve for ER(+), lymphnode negative, Tamoxifen-treated, relapse-free survival (N=471).

These results are consistent with previous studies showing thatmitochondrial activity is functionally upregulated in breast cancermetastatic lesions, within surgically excised lymph nodes, using ahistochemical activity stain that detect mitochondrial complex IV. Inaddition, the inventors previously noted that 16 members of the ATPSgene family, including ATP5F1C (4.64-fold; p=1.14E-05), aretranscriptionally upregulated in human breast cancer cells, relative toadjacent stromal cells, in samples derived from N=28 breast cancerpatients.

Existing GEO DataSets (GSE55470) were used to assess the use ofATP-related genes and OXPHOS genes as transcriptional biomarkers ofbreast cancer circulating tumor cells (CTCs) in patients. FIG. 4A showsa HeatMap of the ATP-ABC gene expression profile in the data set, andinclude a legend. FIG. 4B shows a HeatMap of the OXPHOS gene expressionprofile, based on the same legend in FIG. 4A. Generally, the lighter thecell, the higher the absolute value of the expression. Distinguishingbetween positive and negative fold changes is difficult in black andwhite used in connection with this application. The majority of cellsthe first five columns, for the control blood, are green in the originalHeatMap, indicating a negative fold change in expression. Cells in themajority of the remaining columns are red, indicating a positive foldchange in expression. Overall, the data demonstrate that high ATPcontent in CTCs may be useful as a biomarker, to identify and track CTCsin whole blood, thereby potentially improving cancer diagnosis andpreventing metastatic spread.

FIG. 4C shows the results of a Western blot analysis of MDA-MB-231 cellsin the ATP-high and ATP-low sub-populations. The results show thatmitochondrial markers and CTC markers are both upregulated in ATP-highMDA-MB-231 cells. Mitochondrial markers from Complexes I to V, includingATP5F1C, were all over-expressed in MDA-MB-231 cells in the ATP-highsub-population, relative to MDA-MB-231 cells in the ATP-lowsub-population. In addition, two known markers of CTCs and metastasis(VCAM-1 and Ep-CAM) were over-expressed in MDA-MB-231 cells in theATP-high sub-population. Beta-actin and Beta-tubulin were used asmarkers for equal protein loading.

Taken together, the bioinformatics data and analysis shows thatincreased mitochondrial ATP synthesis could be a key driver of 3Danchorage-independent growth and metastasis. Based on this analysis,cancer cells having the highest levels of ATP would be highlyproliferative, more stem-like, undergo 3D-anchorage independent growthand would possess other aggressive behaviors, as compared to cancercells with lower levels of ATP. Likewise, those cells having the lowestlevels of ATP would be more dormant. Both sub-populations haveconsiderable value for, among other uses, cancer research and drugscreening. The present approach provides methods for separating thesesub-populations from cancer cell populations through metabolicfractionation. Cancer cell populations have numerous sub-populations.CSCs are a small sub-population of cancer cells having self-renewalproperties, are capable of differentiation, and they show tumorigenicitywhen transplanted. As described herein, however, not all CSCs arecreated equal. CSCs separated and purified on the basis of ATP levelshave unique phenotypic properties not found in naturally-occurringcancer cell populations, or even in CSCs separated and purified usingconvention markers such as CD44, CD24, and CD133.

Under the present approach, cells, and preferably cancer cells, may befractionated based on metabolic condition using a fluorescentATP-labeling dye, such as ATP-Red 1, and flow cytometry. ATP levelsultimately determine the phenotypic traits of cancer cells, such as“stemness” and proliferation capacity. The ATP-labeling dye can thus beused to identify and purify the energetically “fittest” cancer cellsfrom within the total cell population. The inventors selected BiotrackerATP-Red 1, a fluorescent vital dye, to label ATP in living cancer cells.It should be appreciated that other fluorescent ATP imaging probes,including later-developed probes, may be used without departing from thepresent approach. Preferably, the fluorescent ATP imaging probe targetsmitochondrial ATP.

ATP-Red 1 is normally non-fluorescent, but becomes fluorescent whenbound to ATP, but not to any other related nucleotides or metabolites,including ADP. More specifically, BioTracker ATP-Red 1 does notrecognize sugars (arabinose, galactose, glucose, fructose, ribose,sorbose, sucrose or xylose) or other nucleotides (AMP, ADP, CMP, CDP,CTP, UMP, UDP, UTP, GMP, GDP or GTP). Importantly, this fluorescent ATPimaging probe exhibits a “turn-on” fluorescence-response toward ATP,with a near 6-fold fluorescence enhancement. Using fluorescencemicroscopy, ATP-Red 1 is predominantly detected within mitochondria, themajor source of cellular ATP production. Therefore, ATP-Red 1 ispreferred as a fluorescent probe to metabolically fractionate the cancercell population by flow cytometry.

The cancer cell population may be separated or fractionated intoATP-high and ATP-low cell sub-populations, and then subjected tophenotypic characterization. The sub-populations may be defined termterms of a percentage of the top and bottom fluorescent signals (e.g.,top and bottom 20%, top and bottom 1%, etc.), and the FACS gate cut-offsfor cell selection and collection are determined based on the selectedpercentages. The data disclosed herein primarily relied on the top andbottom 5%, and the top and bottom 10% as the gate cut-offs, but itshould be appreciated that other percentages may be used withoutdeparting from the present approach. Of course, the percentage should beless than 50%, and it should be expected that the larger the percentage,the less specific the phenotypic characterization will be for a givencell population.

FIG. 5A illustrates an embodiment of the metabolic fractionationprocedure according to the present embodiment. The fluorescent ATPimaging probe may be dissolved in media and incubated 501. The resultsdescribed herein involved 5 μM Biotracker ATP-Red as the fluorescent ATPimaging probe, dissolved in media and incubated in cells for 30 minutes.The cells were then washed with PBS and trypsinized, and re-suspended ina FACS buffer and passed through a 40 μm cell strainer 503. Cellsderived from 3D spheres or 2D adherent condition were analyzed using aFACS sorter instrument (e.g., SONY SH800) 505. Cells were gated at thedesired ATP content, using ATP-based fluorescent signals (e.g.,top/bottom 1%, 2%, 3%, 4%, 5%, 10%, etc.) and sorted 507. FIG. 5Billustrates an example of metabolic fractionation of MCF7 cells withATP-Red 1, to isolate ATP-high (top 5%) and ATP-low (bottom 5%) cellsub-populations. The bulk (5%) population was also selected forcomparison purposes. The right image shows the cell count at variousfluorescent intensities, and identifies the regions of the ATP-high (top5%) sub-population, the ATP-low (bottom 5%) sub-population, and the bulkmedian. The left image of FIG. 5B shows the mean ATP-based fluorescentsignal for each sub-population. Based on mean signal intensity, weestimate that ATP-high MCF7 cells have approximately 15-fold higherlevels of ATP, as compared with the ATP-low population; and 2-foldhigher levels of ATP, as compared with the bulk cell population.

FIGS. 6A and 6B show results from a continuous, real-time assay systemon cell proliferation of all three cell sub-populations (ATP-low 5%,Bulk 5%, ATP-high 5%). Cell proliferation was assessed using thexCELLigence® RTCA DP instrument. Cells were first sorted for ATPcontent, counted and seeded (1×10⁴ in common media) in RTCA DP E-Platesfor real-time growth analysis. Graphically, the 3 sub-populations(ATP-low 5%, Bulk 5%, ATP-high 5%) are all represented. The resultsindicate that the ATP-high population is approximately 2-fold moreproliferative, relative to the bulk cell population and approximately5-fold more proliferative, relative to the ATP-low population, after 120hours. Data represent the mean±SD, n=3. One-way ANOVA, Dunnett'smultiple comparisons test, **p<0.001, ***p<0.001, ****p<0.0001. As canbe seen in FIG. 6A, the ATP-high sub-population had a significantlyhigher cell index compared to the other sub-populations, and the ATP-lowsub-population and a significantly lower cell index compared to theother sub-populations. FIG. 6B shows the slope of the cell index overtime for each sub-population. At each time point (24, 48, 72, 96 and 120hours) the slope of the ATP-high cell population, was significantlyhigher compared to the other 2 sub-populations. Data represent mean±SD,n=3. Two-way ANOVA, Tukey corrected, *p<0.01. It is apparent that theATP-high sub-population had the highest rate of proliferation across theentire 120-hour assessment. The results indicate that the ATP-highpopulation at least, approximately, 2-fold more proliferative, relativeto the bulk cell population, and at least, approximately, 5-fold moreproliferative, relative to the ATP-low population. This demonstratesmitochondrial ATP levels are a key determinant of MCF7 cellproliferation, and that the metabolic fractionation with a fluorescentATP imagine probe of the present approach is an effective technique foridentifying the most proliferative, and least proliferative, cellsub-populations.

Further assays confirmed that the ATP-high MCF7 sub-population areenergetically hyper-proliferative, have significantly increased 3Danchorage-independent growth, cancer stem cell markers, andmitochondrial mass. To confirm the selectivity of ATP-Red 1,Cell-Titer-Glo was used to measure ATP levels in cells, after flowcytometry. However, as Cell-Titer-Glo is a luciferase-based assay, itrequires cell lysis to detect ATP levels and as a consequence, it cannotbe used for live cell sorting or imaging. After cell counting, equalnumbers of single cells were then used to evaluate their relative ATPcontent by luminescence, using the Varioskan™ LUX plate reader. FIG. 7Ais a bar graph that shows cells in the ATP-high MCF7 sub-population haveat least a 15-fold increase in ATP levels, while bulk cells showed abouta 7-fold increase in ATP, relative to the ATP-low cell population. Thisalso shows that the ATP-high sub-population has at least twice the ATPlevel of the bulk cells in the MCF7 population.

The 3D-mammosphere assay was used to measure anchorage-independentgrowth, which is a functional read-out for CSC activity and CSCpropagation. FIG. 7B shows results of the 3D-mammosphere assay for theATP-high, bulk, and ATP-low sub-populations, using 5% as the gatecutoff. FIG. 7C shows comparative images of the cell sub-populationsafter the assay. Images of 3D mammospheres were acquired using the EVOSFL Auto2 microscope. The panels represented the 3 sorted cell populationcells. Representative Images are shown. A 4X objective was used. Scalebar =1,000 μm. The ATP-high MCF7 cell sub-population showed a 9-foldincrease in 3D spheroid formation relative to the ATP-lowsub-population, and nearly double the mammosphere formation of the bulksub-population. These data indicate that ATP-high cells would be betterable to undergo 3D anchorage-independent growth than the bulk CSCpopulation.

Two well-established CSC markers, CD44 and ALDH activity, were used toexamine the “stemness” of the sub-populations. FIG. 7D shows that theATP-high MCF7 cell sub-population (right bars) was enriched nearly4-fold in CD44 cell surface expression and about 5.5-fold inALDH-activity, when using a FACS gating cut-off of 5%, compared to theATP-low sub-population (left bars). Similar results were also obtainedwith MitoTracker-Deep-Red, a well-established marker of mitochondrialmass, which revealed a 3-fold increase in the ATP-high MCF7sub-population compared to the ATP-low sub-population. Mitochondrialmass is a specific marker of stemness in CSCs.

These demonstrate that the metabolic fractionation of the presentapproach enriches the CSC activity in the ATP-high sub-population.Importantly, high ALDH activity is considered to be a biomarker of theEMT (epithelial-mesenchymal transition) in CSCs, whereas CD44 isconsidered to be more of an epithelial CSC marker. So, both epithelialand mesenchymal CSCs are significantly enriched in the ATP-high cellsub-population.

Fluorescent vital probes for anti-oxidant capacity and pluripotency alsoselect for a population of ATP-high MCF7 cells. The effectiveness of theBioTracker ATP-Red 1 imaging probe was compared with several otherfluorescent vital dyes, specifically for ATP-high cell populationselectivity. For this purpose, MCF7 cell 2D monolayers were harvestedwith trypsin and lived-stained with a panel of 5 other fluorescentBioTracker probes for i) anti-oxidant capacity, including cystine uptake(“cysteine-FITC”) and gamma-glutamyl-transpeptidase activity, or GGT;ii) pluripotent stem cells; iii) hypoxia; and iv) senescence(beta-galactosidase activity, or β-Gal). Then, total ATP levels weredetermined using Cell-Titer-Glo, immediately following flow cytometry.

FIG. 7E shows the results of the Cell-Titer-Glo of this analysis,showing the fold change in luminescence of the highest 5% (the right barfor each probe) relative to the lowest 5% (the left bar for each probe).Remarkably, the probes for anti-oxidant capacity (cystine uptake and GGTactivity), as well as pluripotency, all selected for the ATP-highsub-population of MCF7 cells. However, of the additional fluorescentvital probes tested, the BioTracker probe that directly measures theuptake of cystine-FITC, was the most effective at selecting the ATP-highcell sub-population, but it was not as effective as ATP-Red 1 (3-foldvs. 20-fold). Interestingly, high anti-oxidant capacity is known to bestrictly associated with stemness and the drug-resistance phenotype. Thehypoxia probe also positively selected the ATP-high cell sub-population.This may be due to the association between hypoxia and increasedmitochondrial biogenesis. However, the senescence probe(beta-galactosidase activity) did not select for either the ATP-high orthe ATP-low cell population.

FIGS. 8A and 8B show results relating to the metabolic profiling of3D-mammospheres and ATP-high MCF7 cells. The intracellular ATP levels inMCF7 cells, cultured either as 2D monolayers or 3D spheroids, werecompared to better understand the metabolism underlying 3D-anchorageindependent growth. The latter cell population is known to behighly-enriched in CSCs. Metabolite levels in MCF7 cells grown as 2Dadherent monolayers or 3D mammospheres were compared, using Promega kits(Cell-Titer-Glo, GSH/GSSG-Glo, NADP-NADPH-Glo, NAD-NADH-Glo). 2Dmonolayers and 3D mammospheres were first dissociated into single cellswith trypsin, syringed with a 25-gauge needle and passed through a 40-μmcell strainer. After cell counting, equal numbers of single cells werethen used to evaluate their relative luminescence content. Note thatcells derived from 3D mammospheres showed over a 2-fold increase in ATPlevels; a near 2-fold increase in reduced glutathione levels; over a2-fold increase in NADP-NADPH levels and near 1.5-fold increase inNAD-NADH levels, all relative to 2D monolayers. Data represent the meanfold increase over adherent cells±SD, n=4. Unpaired t-test, ** p<0.005,***p<0.0005, ****p<0.0001.

FIG. 8A compares the change in luminescence of 3D spheroids (right bar)to 2D monolayers (adherent, left bar) for probes targeting ATP,GSH/GSSG, NADP-NADPH, and NAD-NADH. Quantitative analysis of MCF7 cellsderived from 3D spheroids showed a 2.3-fold increase in ATP levels,relative to 2D monolayer cells. Approximately 2-fold increases in boththe GSH/GSSG ratio and NADP/H levels were observed, and similar resultswere obtained with NAD/H. These data are consistent with the idea that3D anchorage-independent growth may also require increased anti-oxidantcapacity.

Given the foregoing, an ATP-high sub-population of 2D monolayer cellsare expected to have an ability to undergo 3D anchorage-independentgrowth. Under conditions of low-attachment, >90% of MCF7 cells normallyundergo anoikis, a specialized form of apoptotic cell death. Higher ATPlevels would presumably allow CSCs to better resist the high stress ofgrowth in suspension, caused by the absence of cell-substrateattachment. However, higher energy reserves might also confer resistanceto multiple stressors, resulting in multi-drug resistance.

ATP-high and ATP-low MCF7 cells were subjected to metabolic profilingfor NAD/H and two key anti-oxidants, GSH and NADP/H using Promega kits(Cell-Titer-Glo, GSH/GSSG-Glo, NADP-NADPH-Glo, NAD-NADH-Glo). Cells in2D monolayers were first stained with BioTracker ATP-Red 1 and sorted byATP content by flow cytometry. After cell counting, equal numbers ofsingle cells were then used to evaluate their relative luminescencecontent. Note that ATP-high cells showed a near 25-fold increase in ATPlevels; a 6-fold increase in reduced glutathione levels; a near 8-foldincrease in NADP-NADPH levels and >2-fold increase in NAD-NADH levels,all relative to ATP-low MCF7 cells. Data represent the mean foldincrease over ATP-low 5% cells±SD, n=4. Unpaired t-test, ** p<0.005,***p<0.0005.As shown in FIG. 8B, cells in the ATP-high sub-populationcontain over 1.5-fold more NAD/H, over 7.5-fold more NADP/H, and over7-fold more reduced glutathione (GSH/GSSG ratio), all relative to cellsin the ATP-low sub-population. These data show that MCF7 cells in theATP-high sub-population are more energetic and, as consequence, theyfortify their anti-oxidant capacity. High levels of anti-oxidants areknown to be associated with drug-resistance in cancer cells, indicativeof a multi-drug resistant phenotype. Cells in the ATP-high MCF7sub-population thus mimic the 3D metabolic phenotype, demonstrating thatthe present approach of separating ATP-high cells from a populationproduces a unique phenotype, having numerous potential uses.

The ATP-high and ATP-low sub-population phenotypes exist across numerouscancer types. ATP-high sub-populations of MCF7, T47D, MDA-MB-231 andMDA-MB-468 cells all show increased 3D anchorage-independent growth.Compositions of ATP-high and ATP-low cell sub-populations from threeother human breast cancer cells lines, T47D, MDA-MB-231 and MDA-MB-468cells, prepared with a FACS gating cut-off of 10%. The relative amountof ATP in the ATP-high and ATP-low cell sub-populations, wasindependently validated using Cell-Titer-Glo. 2D monolayer cells werefirst stained with BioTracker ATP-Red 1 and sorted by ATP content, usinga flow cytometer. After cell counting, equal numbers of single cellswere then used to evaluate their relative luminescence content. In thisseries of experiments, we used a cut-off of 10% to define the ATP-highand ATP-low cell populations. Note that this metabolic fractionationscheme can be successfully applied to other breast cancer cell lines.Data represent the mean fold increase over ATP-low 10% cells±SD, n =3.Unpaired t-test, * p<0.05, ** p<0.005, ***p<0.0005.

FIGS. 9A and 9B show Cell-Titer-Glo and 3D mammosphere formation resultsfor ATP-high and ATP-low sub-populations of MCF7, T47D, MDA-MB-231 andMDA-MB-468 cells, using a 10% gate. FIG. 9A illustrates that theATP-high sub-populations of all these cell lines showed increases in ATPcharacteristic of the ATP-high sub-population phenotype, as confirmedusing the luciferase-based Cell-Titer-Glo assay, with a 2-to-3-foldincrease in total ATP levels. As seen in FIG. 9B, similar results wereobtained with the 3D spheroid assay, indicative of an increase in CSCactivity and propagation between 1.75- and 3-fold, depending on the cellline examined. Cells in 2D monolayers were first stained with BioTrackerATP-Red 1 and sorted by ATP content, using a flow cytometer. After cellcounting, 5×10³ cells were seeded onto poly-HEMA coated 6-well plate andcounted after 5 days. Note that the ATP-high cell population of MCF7,T47D, MDA-MB-231 and MDA-MB-468 cells all showed an increased capacityfor 3D anchorage-independent growth. Data represent the mean foldincrease over ATP-low 10% cells±SD, n=3. Unpaired t-test, *** p<0.0005,****p<0.0001.

Cells in the ATP-high sub-populations show increases in oxidativemitochondrial metabolism, glycolytic rates and cell cycle progression.FIG. 10A shows luminescence in ATP-high and ATP-low sub-populations(10%) in a MCF7 cell population after a 24-hour period. After cellcounting, equal numbers of single cells were then used to evaluate theirrelative ATP content by luminescence using the Varioskan™ LUX platereader, 24 hours after plating. For these experiments, which requiredlarger numbers of cells, a cut-off of 10% was used to define theATP-high and ATP-low cell populations. Data represent the mean foldincrease±SD over ATP-low 10% cells, n=3. Unpaired t-test,****p<0.0001.The observed increases in ATP levels were reduced to 3-foldby 24 hours after plating the ATP-high cells as a 2D monolayer,indicating that the highly energetic, ATP-high phenotype is relativelytransient, consistent with a more stem-like phenotype.

FIGS. 10B through 10E show the results of metabolic flux analysis on theATP-high and ATP-low sub-populations. The OCR (oxygen-consumption rate)was determined using the Seahorse XFe96, via metabolic flux analysis.Note that the ATP-high MCF7 cell population shows an increase in bothbasal and maximal respiration, as well as mitochondrial ATP-production.Cell populations were analyzed 24 hours after plating. Data representthe % fold increase±SD over ATP-low 10% cells, n=3. Unpaired t-test, *p<0.05, ** p<0.005. The ECAR (extracellular acidification rate) wasdetermined using the Seahorse XFe96, via metabolic flux analysis. Notethat the ATP-high MCF7 cell population shows an increase in glycolysis.Cell populations were analyzed 24 hours after plating. Data representthe % average fold increase±SD over ATP-low 10% cells, n=3. Unpairedt-test, ns=not significant, *** p<0.0005. The energetic profiles inFIGS. 10B and 10C show that the ATP-high sub-population is metabolicallyactive relative to the ATP-low population. Following 24 hours after cellattachment, ATP-high MCF7 cell monolayers showed a 2-fold increase inbasal respiration, a 1.5-fold increase in maximal respiration and a3-fold increase in ATP production. Similarly, ATP-high MCF7 monolayercells also showed a 1.5-fold increase in basal glycolytic rate. Theglycolytic rates in FIGS. 10D and 10E demonstrate that the ATP-highsub-population is significantly more bioenergetic than the ATP-lowsub-population.

An evaluation of the proliferative capacity of the ATP-high cellsub-population reveals that the ATP-high cell sub-populations arestrikingly more proliferative than the ATP-low sub-populations, in avariety of cancer types. FIGS. 11A-11D show cell cycle progression inMCF7, T47D, MDA-MB-468, and MDA-MB-231 cells, using FACS analysis withpropidium iodide to detect DNA-content. As can be seen, the ATP-highcell sub-populations were strikingly more proliferative than theATP-low, with a shift from the G0/G1-phase to the S-phase and theG2/M-phase. More specifically, the G0/G1-phase was reduced fromapproximately 80-88% to 60-64%, while the S-phase was increased from4-8% to 9-21%. Similarly, the G2/M-phase was increased, from 7-12% to17-30%. These clear increases in cell cycle progression in the ATP-highcell sub-population, relative to the ATP-low sub-population, wereobserved in all 4 cell lines. Overall, this represents a 1.9- to3.6-fold increase in the number of cells in S-phase and a 1.8- to3.8-fold increase in the number of cells in the G2/M-phase, across the 4cell lines tested. Interestingly, the largest increase in S-phase wasobserved in MCF7 cells, while the largest increase in the G2/M-phase wasobserved in MDA-MB-231 cells.

Conversely, the ATP-low population in each cell line was essentiallyquiescent, with 80-88% of the cells in the G0/G1-phase of the cellcycle, demonstrating a predominant phenotype of cell cycle arrest. Thus,the ATP-low cell sub-population fits well with the current definition ofcancer cell dormancy.

Therefore, high ATP levels are a primary determinant of “stemness”traits, anchorage independent growth, and cell proliferation. As such,the practical approach described herein allows for successfullyisolating the bioenergetically “fittest” and most proliferative cancercells, from the total cell population, and forming a new composition ofcells having unique phenotypic properties. These properties haveimplications for drug-resistance.

MCF7 cells in the ATP-high subpopulation show a multi-drug resistancephenotype. The 3D-mammosphere assay was used to explore the differentialsensitivity of ATP-high and ATP-low MCF7 cell sub-populations to fourdifferent classes of drugs, using as a functional readout ofdrug-resistance. The drug classes include Tamoxifen, doxycycline, DPI,and Palbociclib. FIG. 12A shows results for the ATP-low sub-population(bottom 5%), and FIG. 12B shows results for the ATP-high subpopulation(top 5%). Two concentrations for each drug class are shown in FIGS. 12Aand 12B.

Tamoxifen is an FDA-approved drug routinely used to clinically targetER(+) breast cancer cells, that often leads to Tamoxifen-resistance andtreatment failure, resulting in tumor recurrence and distant metastasis.Interestingly, 3D-mammosphere formation by ATP-low MCF7 cells wasremarkably sensitive to Tamoxifen treatment, resulting in a reduction by˜40% at 1μM and by >90% at 5 μM. In contrast, FIG. 12B shows that3D-mammosphere formation by ATP-high MCF7 cells was strikingly resistantto Tamoxifen, as 3D-mammosphere formation remained high at 5 μM,representing >80% of the vehicle-treated control levels. Thus, ATP-highMCF7 cells are clearly Tamoxifen-resistant.

FIG. 12B shows that ATP-high MCF7 cells were also resistant to amitochondrial OXPHOS inhibitor, namely diphenyleneiodonium (DPI). Forexample, DPI treatment of ATP-low cells reduced 3D-mammosphereformationby >90% at 100 nM. On the other hand, DPI treatment (100 nM) of ATP-highcells only reduced 3D-mammosphere formation by ˜55%. Therefore, bothsub-populations were sensitive to a mitochondrial inhibitor, butATP-high cells were clearly more resistant.

Doxycycline is an FDA-approved antibiotic which behaves as an inhibitorof mitochondrial ribosome translation. Comparing FIG. 12A to FIG. 12Bshows that the ATP-high sub-population was largely resistant toDoxycycline, at concentrations that were highly effective in ATP-lowMCF7 cells, namely 25 μM and 50 μM.

The efficacy of Palbociclib, an FDA-approved CDK4/6 inhibitor, is alsoevident in FIGS. 12A and 12B. Palbociclib treatment of ATP-low cellsreduced 3D-mammosphere formation by ˜75% at 12.5 nM. However,Palbociclib treatment (12.5 nM) of ATP-high cells only reduced 3Dspheroid formation by -50%. As such, ATP-high cells were also moreresistant to a CDK4/6 inhibitor. As can be seen, the ATP-highsub-population is a phenotype having resistance to several drug types.

Biotracker-ATP-Red 1 was compared with well-established markers ofstemness, CD44, and ALDH-activity. In order to directly compare theeffectiveness of ATP-Red 1 with other CSC markers, a double-labelingstrategy was applied to both MCF7 and MDA-MB-231 cells. The cells weredouble-labeled for CD44 and ATP, using different fluorescent channelsfor detection. In the case of CD44 and ATP, this resulted in 4experimental groups: CD44-high/ATP-high, CD44-high/ATP-low,CD44-low/ATP-high, and CD44-low/ATP-low.

After cell sorting, the resulting four sub-populations were thensubjected to the 3D-mammosphere assays, as a functional read-out ofstemness. ATP versus CD44 cell surface expression. 3Danchorage-independent growth was measured in the different cellsub-populations, as a functional readout of stemness, using both MCF7and MDA-MB-231 lines, after cell sorting. Briefly, 2D-monolayers werefirst co-stained with both BioTracker-ATP (PE channel) and Anti-CD44(APC-channel) and subjected to flow cytometry, using the SONY SH800 cellsorter. After cell counting, 5×10³ cells were seeded in poly-HEMA coated6-well plates and 3D-mammospheres were counted 5 days after plating. Asshown in FIGS. 13A and 13B, CD44-low/ATP-low cells showed the leastanchorage-independent growth, as expected given the phenotypicproperties of these sub-populations. Therefore, CD44-low/ATP-low cellswere chosen as the point for normalization. Two cell sub-populationsshowed the most anchorage independent growth: CD44-high/ATP-high andCD44-low/ATP-high. Therefore, high levels of ATP are the dominantdeterminant of stemness, as compared with CD44, in both MCF7 andMDA-MB-231 cells.

Considering only the CD44-high population and double-labeling with ATPallowed for separating the CD44-high population into 2 sub-populations,one with high capacity for propagation (CD44-high/ATP-high) and one withlow capacity for propagation (CD44-high/ATP-low). Therefore, theCD44-high/ATP-low population clearly showed significantly lessanchorage-independent growth and represents a more “dormant”sub-population of CD44(+) CSCs.

Virtually identical results were also obtained by double-labeling withADLH-activity and ATP. 3D anchorage-independent growth was measured inthe different cell sub-populations, as a functional readout of stemness,using both MCF7 and MDA-MB-231 lines, after cell sorting. Briefly, 2Dmonolayers were first co-stained with both BioTracker-ATP (PE channel)and for ALDH-activity (APC-channel) and subjected to flow cytometry,using the SONY SH800 cell sorter. After cell counting, 5×10³ cells wereseeded in poly-HEMA coated 6-well plates and 3D mammospheres werecounted 5 days after plating. FIGS. 13C and 13D show results of themammosphere formation assay for MCF7 and MDA-MB-231 cell lines,respectively, double-labeled for ALDH-activity and ATP. As expected, thetwo cell populations that showed the most anchorage-independent growthwere ALDH-high/ATP-high and ALDH-low/ATP-high. Therefore, high levels ofATP are the dominant determinant of stemness, as compared with ALDH, inboth MCF7 and MDA-MB-231 cells. Similarly, double-labeling with ATPallows for the separation of the ALDH-high population into 2sub-populations, one with high capacity for propagation(ALDH-high/ATP-high) and one with low capacity for propagation(ALDH-high/ATP-low).

The foregoing demonstrates that the present approach is a powerful andeffective approach for sub-fractionating CSCs into a more active,hyper-proliferative sub-population, and a more dormant sub-population,using ATP as a secondary marker for dormancy. The results also indicatethat ATP levels are a functional regulator of dormancy in CSCs.

The role of mitochondrial ATP in cell migration, invasion andspontaneous metastasis was also explored. The data demonstrate thatmitochondrial ATP is an energetic biomarker for the process of cancercell metastasis. MDA-MB-231 cells are a well-established model for thestudy of cell motility and metastasis, both in vitro and in vivo. Theability of ATP-high and ATP-low subpopulations of MDA-MB-231 cells toundergo cell migration and invasion were evaluated by employing amodified Boyden chamber assay, using Transwells. The bulk (5%)population was also selected for comparison purposes. To study invasion,the Transwells were coated with extracellular matrix, namely Matrigel,to prevent simple cell migration. For both cell migration and invasionassays, serum was used as a chemoattractant. Migration and invasionparameters were independently quantitated, using both crystal violetstaining intensity and cell number.

FIGS. 14A and 14B show the results of this migration and invasionanalysis. The ATP-high MDA-MB-231 cells showed a 20- to 40-fold increasein their ability to undergo cell migration, relative to ATP-low cells.As expected bulk (5%) cells showed an intermediate phenotype. ATP-highMDA-MB-231 cells showed a 15- to 25-fold increase in their ability toundergo invasion, relative to ATP-low cell population. As such, ATP-highMDA-MB-231 cells represent the pro-metastatic cell sub-population invivo.

For further evaluation, a well-established in vivo metastasis assay,involving the chorio-allantoic membrane (CAM) in chicken eggs, was usedto quantitatively measure spontaneous metastasis. After cell sorting toisolate ATP-high and ATP-low cell sub-populations, an inoculum of 30,000cells (MDA-MB-231) was added onto the CAM of each egg (day E9) and theneggs were randomized into groups. On day E18, the lower CAM wascollected to evaluate the number of metastatic cells, as analyzed byqPCR with specific primers for Human Alu sequences. Non-injected eggswere also evaluated in parallel, as a negative control for specificity.Greater than 20 eggs were processed for each experimental condition.

FIG. 15 shows the results of the spontaneous metastasis in vivo CAMassay. The data illustrate that MDA-MB-231 cells in the ATP-highsub-population were 4.5-fold more metastatic than ATP-low cellsub-population. These sub-populations were derived from the same cellline. Therefore, MDA-MB-231 cells in the ATP-high sub-populationrepresent the pro-metastatic CSC sub-population. As discussed above inconnection with FIG. 4C, MDA-MB-231 cells in the ATP-high sub-populationalso over-express two CTC and metastasis markers (VCAM-1 and Ep-CAM),indicating that the hyper-proliferative CSCs are the CTCs responsiblefor seeding distant metastasis.

The present approach can therefore be used to detect the potential of acancer to metastasize. For example, a biological sample from a cancermay be metabolically fractionated to assess the content of the ATP-highsub-population, and that content may be used to estimate the likelihoodof the cancer to metastasize. Early detection and analysis of cells in acancer patient's ATP-high sub-population provides invaluableopportunities to diagnose the risk of metastasis and identify anappropriate treatment, such as with an ATP-depletion therapeutic asdiscussed herein.

The Tempo-ATP protein-biosensor to purify ATP-high MCF7 cells providesindependent validation of the use of ATP as a new biomarker for stemnessin cancer cells. Tempo-ATP, a fluorescent protein-biosensor, is acompletely different probe for detecting ATP levels in living cells, andwas used for detecting high and low levels of ATP.

Tempo-ATP-MCF7 cells, recombinantly over-expressing a cytosolicfluorescent protein ATP-biosensor, were custom-generated byTempo-Bioscience, Inc. (San Francisco, Calif., USA), using apuromycin-resistance marker for cell selection. This protein-basedfluorescent ATP-biosensor has an excitation wavelength of 517-519-nm andan emission of 535-nm. It consists of an ATP-binding peptide, fusedin-frame with a GFP-like fluorescent reporter protein. TheTempo-ATP-MCF7 cells were sorted for GFP content, as a surrogate markerfor cytoplasmic ATP-content, using a flow cytometer(Excitation=517-519-nm; Emission=535-nm). After cell counting, equalnumbers of single cells were then used to evaluate their metabolic andphenotypic behavior.

The results are shown in FIGS. 16A-16C. The relative increase inluminescence of the GFP-high sub-population, relative to the GFP-lowsub-population, is shown in FIG. 16A. Cell cycle progression data aresummarized in FIG. 16B, and mammosphere formation assay results areshown in FIG. 16C. As expected based on the discussion thus far, theATP-high Tempo-MCF7 cells showed significant increases in ATP, morereduced glutathione, NADP/H and NAD/H, as well as increases in cellcycle progression and 3D anchorage-independent growth. The Tempo-ATPdata independently validation the BioTracker ATP-Red 1 results, andspecifically, that high-ATP levels are a key determinant of anti-oxidantcapacity, cell proliferation, and 3D anchorage-independent growth.Although Tempo-ATP was effective, BioTracker ATP-Red 1 was significantlymore effective, because of its direct localization within mitochondria,the main cellular source ATP production.

The present approach demonstrates that high ATP production is a keydriver of “stemness” traits and proliferation in cancer cells. Theobservations disclosed herein could explain the molecular basis ofmetabolic heterogeneity observed in the cancer cell population, as wellas its relationship to phenotypic behaviors, such as i) rapid cell cycleprogression and ii) anchorage-independent growth, which are bothrequired for the metastatic dissemination of CSCs in vivo.

As demonstrated, ATP may be used as a biomarker to metabolicallyfractionate a cancer cell population, and identify hyper-prolific anddormant sub-populations. This, in turn, indicates that ATP-depletiontherapy may be effective for treating the hyper-prolificsub-populations, and reduce or eliminate the likelihood of tumorrecurrence and metastasis.

Under the present approach, a vital fluorescent dye that allows one tomeasure ATP levels in living cells, such as BioTracker ATP-Red 1, may beused as an imaging probe for metabolic fractionation. More specifically,BioTracker ATP-Red 1 staining may be coupled with a bioenergeticfractionation scheme, in which the total cell population is subjected toflow cytometry, to isolate the ATP-high and ATP-low sub-populations ofthe population. MCF7 cells, an ER(+) human breast cancer cell line, wereused in many of the examples discussed above, but it should beappreciated that the present approach may be used for any cell line, andany cancer type. The metabolic fractionation approach allows forisolating the most “energetic” cancer cells within the total cellpopulation. Advantageously, the resulting ATP-high cancer cellsub-population may be targeted for eradication via ATP-depletiontherapy, and serve as a basis for drug discovery and development. Giventhe phenotypic properties, the ATP-high sub-population may also be usedfor evaluating therapies to prevent or reduce the likelihood ofrecurrence and metastasis.

In a parallel line of research, the inventors identified over 20mitochondrially-targeted therapeutics that could be used to effectivelyachieve ATP-depletion therapy. These potential therapeutics include:FDA-approved drugs (Doxycycline, Tigecycline, Azithromycin, Pyrviniumpamoate, Atovaquone, Bedaquiline, Niclosamide, Irinotecan); naturalproducts/nutraceuticals (Actinonin, CAPE, Berberine, Brutieridin,Melitidin); and experimental compounds (Oligomycin, AR-C155858,Mitoriboscins (see International Application No. PCT/US2018/022403,filed Mar. 14, 2018, and incorporated by reference in its entirety.),Mitoketoscins (see International Application PCT/US2018/039354, filedJun. 25, 2018, and incorporated by reference in its entirety),Mitoflavoscins (see International Patent Application PCT/US2018/057093,filed Oct. 23, 2018 and incorporated by reference in its entirety.), TPPderivatives (including Dodecyl-TPP and 2-Butene-1,4-bis-TPP, seeInternational Patent Application PCT/US2018/062174, filed Nov. 21, 2018and incorporated by reference in its entirety.)). A triple-combinationof two antibiotics together with Vitamin C (Doxycycline, Azithromycinand Ascorbic acid) was found to be particularly potent for targetingmitochondria, inducing ATP-depletion and inhibiting CSC propagation, atsub-antimicrobial levels (see International Patent ApplicationPCT/US2019/066541, filed Dec. 16, 2019 and incorporated by reference inits entirety). The ATP-depletion compound may be an existing compoundmodified to increase efficacy, cell membrane penetration, and/ormitochondrial uptake, such as those described in International PatentApplication PCT/US2018/033466, filed May 18, 2018 and incorporated byreference in its entirety, and International Patent ApplicationPCT/US2018/062956, filed Nov. 29, 2018 and incorporated by reference inits entirety. For example, Doxycycline conjugated with a fatty acid,such as Myristate, may be used as an ATP-depletion compound. In someinstances, it may be appropriate to administer an increased dose of acompound, such as when the ATP-high sub-population shows resistance tothe compound at a dose normally prescribed in the art. A compound may beadministered in It should be appreciated that any of the foregoingcompounds may be used as an ATP-depletion therapeutic, to target theATP-high sub-population, and prevent or reduce the likelihood ofrecurrence and metastasis. It should also be appreciated that any of theforegoing compounds may be used as a therapeutic agent to beadministered to a cancer patient when the expression levels of the5-member gene signature of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB, ina biologic sample of the patient's cancer, are found to be elevatedrelative to expression levels in a non-cancerous biologic sample fromthe patient.

As many of the compounds are repurposed FDA-approved antibiotics, withexcellent safety profiles, Phase II clinical trials are warranted. Forexample, a Phase II clinical pilot study of Doxycycline has alreadyshown that this over 50-year-old antibiotic is indeed effective inmetabolically targeting the CSC population in early breast cancerpatients, as demonstrated using CD44 and ALDH1 as specific CSC markers.Mitochondrial ATP-depletion therapy is expected to functionally mimicfasting and/or caloric restriction, thereby more effectively starvingCSCs to death. Under the present approach, fasting and/or caloricrestriction may be included as part of anti-cancer therapy, to increasethe effectiveness of an ATP-depletion therapy. For example, a patientreceiving ATP-depletion therapy may fast for a period such as 12, 16,24, 36, or 48 hours, before receiving administration of a therapeuticcompound, and/or may fast for a period such as 12, 16, 24, 36, or 48hours, after receiving administration of the therapeutic compound. Insome embodiments, the fast may take place before and afteradministration of the therapeutic compound, to increase theATP-depletion effect. This has important implications for cancerprevention and for potentially extending human lifespan during aging.

Cells in the ATP-high sub-populations show a multi-drug resistantphenotype, with enhanced anti-oxidant capacity. Previous studies haveshown that high anti-oxidant capacity, due to increased levels ofreduced glutathione, elevated NADPH, and activated NRF2 signaling,significantly contributes to the onset of multi-drug resistance. MCF7cells in the ATP-high sub-population have an increased anti-oxidantcapacity, with elevated levels of reduced glutathione, and areintrinsically resistant to four different classes of drugs (Tamoxifen,Palbociclib, Doxycycline and DPI). Therefore, the existence of theATP-high CSC phenotype may help to mechanistically explain thepathogenesis of multi-drug resistance, during cancer therapy. In thiscontext, current cancer therapy may allow only the metabolically“fittest” cancer cells to survive. Those cells, in turn, present thegreatest risk of recurrence and metastasis.

The data disclosed above also show a direct causal relationship betweenmitochondrial “power” and Tamoxifen-resistance. For example, MCF7-TAMRcells that were generated via chronic exposure to increasingconcentrations of Tamoxifen, resulting in Tamoxifen-resistance, showedelevated levels of mitochondrial OXPHOS and ATP production. In MCF7-TAMRcells, acquired Tamoxifen-resistance was due to the over-expression oftwo key anti-oxidant proteins (NQO1 and GCLC) and their positivemetabolic effects on mitochondrial metabolism, as revealed by unbiasedproteomics analysis. In addition, recombinant over-expression of eitherNQO1 or GCLC in MCF7 cells autonomously conferred about a 2-foldincrease in mitochondrial ATP-production and Tamoxifen-resistance.Moreover, recombinant over-expression of a somatic mutation (Y537S) inthe estrogen receptor (ER-alpha; ESR1), clinically associated withacquired Tamoxifen-resistance in breast cancer patients, geneticallyconferred elevated mitochondrial biogenesis, OXPHOS and high ATPproduction. The proteomic profiles of MCF7-TAMR cells andMCF7-ESR1(Y537S) cells also showed considerable overlap in thebiological processes that were functionally activated. Finally, 60 geneproducts functionally-associated with mitochondrial ATP production, werepredictive of Tamoxifen-resistance in ER(+)/Luminal A breast cancerpatients. These predictive biomarkers included 18 differentmitochondrial ribosomal proteins (MRPs) and over 20 distinct componentsof the mitochondrial OXPHOS complexes. The data disclosed herein showthat “naïve” MCF7 cells in the ATP-high sub-population are intrinsicallyresistant to Tamoxifen, without any prior exposure to Tamoxifen. Thishas important clinical implications for optimizing the effectiveness ofhormonal breast cancer therapy.

It has been previously reported that treatment with conventionalchemotherapeutic regimens actually increases the number of CSCs, whileselectively killing “bulk” cancer cells. Until this disclosure, nometabolic hypotheses have been proposed to explain the phenomenon. Chanand colleagues (from Genentech, Inc.) examined the effects ofgemcitabine and etoposide on the total cancer cell population.Remarkably, they observed that after treatment with gemcitabine andetoposide, the population of surviving cells showed an increase in ATPcontent, elevated mitochondrial mass, with more mitochondrialrespiration. However, they did not propose a mechanistic explanation forthese observations, nor did they consider the CSC population. Instead,they simply concluded that measuring ATP is not a good read-out toassess the effectiveness of chemo-therapeutic agents. Given the datadisclosed herein, an alternate interpretation of their results is thatgemcitabine and etoposide selectively killed the ATP-low and bulksub-populations of cancer cells, thereby enriching the “energetic”ATP-high sub-population, which are more stem-like and drug-resistant.Therefore, new drug discovery should be initiated to help eradicate theATP-high sub-population of cancer cells.

Higher intracellular ATP levels have also been suggested to account foracquired drug-resistance to oxaliplatin and cisplatin, in a variety ofchronically-treated colon and ovarian cancer cell lines (HT29, HCT116,A2780), although a diverse number of mechanisms have been proposed,including increased glycolysis and/or mitochondrial metabolism. However,in previous studies, ATP levels were measured only after chronicallyselecting for the drug resistant cell population. Therefore, a directcause-effect relationship between ATP production and drug resistancecould not be established.

Previously, the inventors used a more indirect method to isolate“energetic” cancer stem cells (e-CSCS), which employed auto-fluorescenceto detect intracellular FAD, FMN and riboflavin content. SeeInternational Patent Application PCT/US2019/037860, filed Jun. 19, 2019,which is incorporated by reference in its entirety. However, the use ofATP-Red 1 is a direct method and is a substantial improvement. Forexample, the use of high auto-fluorescence (AF; top 5%) to fractionateMCF7 2D monolayers resulted in an AF-high population of cells, with a1.5-fold increase in anchorage independent growth and a near 2-foldincrease in ATP production. In contrast, in the present approach, theuse of ATP-Red 1 (top 5%) resulted in a sub-population of ATP-high MCF7monolayer cells having a 9-fold increase in anchorage independent growthand over a 15-fold increase in ATP content. The ATP-high sub-populationsfrom other cancer cell lines (T47D, MDA-MB-231 and MDA-MB-468) showedsimilarly hyper-proliferative characteristics. Therefore, the use of ATPas a direct energetic biomarker is far superior to auto-fluorescence. Inaddition, ATP-Red 1 was also effective for metabolically fractionatingthe three other breast cancer cell lines tested.

According to the conventional view of tumor dormancy, dormant cancercells undergo slower rates of cell proliferation and/or cell cyclearrest (quiescence), to avoid therapy-induced cell death, leading tomulti-drug resistance. The data disclosed herein show just the opposite:MCF7 cells in the ATP-low sub-population were less proliferative, withover 87% of the cells in the GO/G1 phase of the cell cycle, but weremore sensitive to 4 different classes of drugs, using the 3D-mammosphereassay as a readout. Conversely, MCF7 cells in the ATP-highsub-population were significantly more proliferative, with over 38% ofthe cells in either S-phase or G2/M, showing a clear multi-drugresistance phenotype. Therefore, high levels of mitochondrial ATP are akey driver of both cell proliferation and drug-resistance, as theyrepresent the energetically “fittest” population of cancer cells.

The inventors have shown that treatments with a panel of distinctanti-mitochondrial therapeutics i) metabolically induce ATP-depletionand ii) are sufficient to potently inhibit cancer cell metastasis, usingan in vivo xenograft animal model. These results indicate that high ATPlevels are critical for the processes of CSC metastasis, and areconsistent with the data disclosed herein, showing that that ATP-highCSCs are hyper-proliferative, stem-like, anchorage-independent, withincreases in anti-oxidant capacity and intrinsic multi-drug resistance.Therefore, the ATP-high CSC that may be isolated using the presentapproach is likely responsible for tumor recurrence and metastasis invivo.

The bioinformatic analysis described above shows that ATP-related genesare closely associated with stemness, proliferation and metastasis,especially ATP5F1C, which encodes the gamma-subunit of the catalyticcore of the mitochondrial ATP synthase. Moreover, ATP5F1C is aprognostic biomarker of tumor recurrence and distant metastasis, as wellas a marker of treatment failure in ER(+) patients undergoing Tamoxifentherapy. Also, ATP-high MDA-MB-231 cells showed dramatic increases intheir capacity to undergo both cell migration and invasion in vitro, aswell as spontaneous metastasis in vivo. Mitochondrial ATP, then, plays acritical role in metastatic dissemination. As such, inhibitors ofmitochondrial ATP synthesis should be effective as potentialtherapeutics for conveying metastasis prophylaxis, for eradicating theCSCs in the ATP-high sub-population.

Pharmaceutical compositions of the present approach include anATP-depleting compound (as identified above) in any pharmaceuticallyacceptable carrier. If a solution is desired, water may be the carrierof choice for water-soluble compounds or salts. With respect to watersolubility, organic vehicles, such as glycerol, propylene glycol,polyethylene glycol, or mixtures thereof, can be suitable. Additionally,methods of increasing water solubility may be used without departingfrom the present approach. In the latter instance, the organic vehiclecan contain a substantial amount of water. The solution in eitherinstance can then be sterilized in a suitable manner known to those inthe art, and for illustration by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized. Embodiments including a second inhibitor compound, suchas a glycolysis inhibitor or an OXPHOS inhibitor, may co-administer aform of the second inhibitor available in the art. The present approachis not intended to be limited to a particular form of administration,unless otherwise stated.

In addition to the ATP-depleting compound, pharmaceutical formulationsof the present approach can contain other additives known in the art.For example, some embodiments may include pH-adjusting agents, such asacids (e.g., hydrochloric acid), and bases or buffers (e.g., sodiumacetate, sodium borate, sodium citrate, sodium gluconate, sodiumlactate, and sodium phosphate). Some embodiments may includeantimicrobial preservatives, such as methylparaben, propylparaben, andbenzyl alcohol. An antimicrobial preservative is often included when theformulation is placed in a vial designed for multi-dose use. Thepharmaceutical formulations described herein can be lyophilized usingtechniques well known in the art.

In embodiments involving oral administration of an ATP-depletingcompound, the pharmaceutical composition can take the form of capsules,tablets, pills, powders, solutions, suspensions, and the like. Tabletscontaining various excipients such as sodium citrate, calcium carbonateand calcium phosphate may be employed along with various disintegrantssuch as starch (e.g., potato or tapioca starch) and certain complexsilicates, together with binding agents such as polyvinylpyrrolidone,sucrose, gelatin and acacia. Additionally, lubricating agents such asmagnesium stearate, sodium lauryl sulfate, and talc may be included fortableting purposes. Solid compositions of a similar type may be employedas fillers in soft and hard-filled gelatin capsules. Materials in thisconnection also include lactose or milk sugar, as well as high molecularweight polyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the compounds of the presentlydisclosed subject matter can be combined with various sweetening agents,flavoring agents, coloring agents, emulsifying agents and/or suspendingagents, as well as such diluents as water, ethanol, propylene glycol,glycerin and various like combinations thereof. In embodiments having acarbocyanine compound with a second inhibitor compound, the secondinhibitor compound may be administered in a separate form, withoutlimitation to the form of the carbocyanine compound.

Additional embodiments provided herein include liposomal formulations ofan ATP-depleting compound disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

With respect to pharmaceutical compositions, the pharmaceuticallyeffective amount of an ATP-depleting compound herein will be determinedby the health care practitioner, and will depend on the condition, sizeand age of the patient, as well as the route of delivery. In onenon-limited embodiment, a dosage from about 0.1 to about 200 mg/kg hastherapeutic efficacy, wherein the weight ratio is the weight of theATP-depleting compound, including the cases where a salt is employed, tothe weight of the subject. In some embodiments, the dosage can be theamount of compound needed to provide a serum concentration of the activecompound of up to between about 1 and 5, 10, 20, 30, or 40 μM. In someembodiments, a dosage from about 1 mg/kg to about 10, and in someembodiments about 10 mg/kg to about 50 mg/kg, can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection. In some embodiments, dosagescan be from about 1 μmol/kg to about 50 μmol/kg, or, optionally, betweenabout 22 μmol/kg and about 33 μmol/kg of the compound for intravenous ororal administration. An oral dosage form can include any appropriateamount of active material, including for example from 5 mg to, 50, 100,200, or 500 mg per tablet or other solid dosage form.

The following paragraphs describe the materials and methods used inconnection with the data and embodiments set forth herein. It should beappreciated that those having an ordinary level of skill in the art mayuse alternative materials and methods generally accepted in the art,without deviating from the present approach.

Cell lines and Reagents: ER(+) [MCF7 and T47D] and triple-negative[MDA-MB-231 and MDA-MB-468] human breast cancer cell lines werepurchased from the American Type Culture Collection (ATCC). ATP-Red 1(also known as BioTracker™ ATP-Red Live Cell Dye; #SCT045) was obtainedcommercially from Sigma-Aldrich, Inc.

The HeatMap of FIG. 1A was prepared using the GSE36953 GEO DataSet,previously deposited in the NCBI database. Total RNA was prepared fromMDA-MB-231 cells, a TNBC cell line, under three different growthconditions: 2D-adherent growth, 3D-anchorage-independent growth and invivo tumor growth. Analysis was performed with the Affymetrix HumanGenome U133 Plus 2.0 Array. The HeatMap was generated with QIAGENOmicSoft Suite Software. ATP-related genes were transcriptionallyupregulated under both 3D growth conditions (anchorage-independent andin vivo tumors), all relative to 2D-adherent growth

Flow Cytometry after Vital Staining with ATP-Red 1: Human breast cancercell lines were first grown either as a 2D-monolayer or as 3D-spheroids.Then the cells were collected and dissociated into a single-cellsuspension, prior to analysis or sorting by flow-cytometry with a SONYSH800 Cell Sorter. Briefly, ATP-high and ATP-low sub-populations ofcells were isolated after vital staining with the probe ATP-Red 1. TheATP-high and ATP-low cell sub-populations were selected by gating,within the ATP-Red 1 signal. Unless otherwise stated, cells with thelowest (bottom 5% or 10%) fluorescent signal, or the highest (top 5% or10%) fluorescent signal, were collected as ATP-low and ATP-high,respectively. The cells outside the gates were discarded during sorting,due to the gate settings. However, such settings are often required toensure high-purity during sorting. Data were analyzed with FlowJo 10.1software.

ATP assay with Cell-Titer-Glo: Cell-Titer-Glo (#G7570) was obtained fromPromega, Inc., and was used according to the manufacturer'srecommendations, to measure ATP levels in lysed cells. Cell-Titer-Glo isa luciferase-based assay system.

3D Anchorage Independent Growth Assay: A single cell suspension wasprepared using enzymatic (lx Trypsin-EDTA, Sigma Aldrich, cat. #T3924),and manual disaggregation (25 gauge needle). Five thousand cells wereplated with in mammosphere medium (DMEM-F12/B27/20ng/m1 EGF/PenStrep),under non-adherent conditions, in six wells plates coated with2-hydroxyethylmethacrylate (poly-HEMA, Sigma, cat. #P3932). Cells weregrown for 5 days and maintained in a humidified incubator at 37° C. atan atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days, 3Dspheroids with a diameter greater than 50 μm were counted using amicroscope, fitted with a graticule eye-piece, and the percentage ofcells which formed spheroids was calculated and normalized to one(1=100% MFE; mammosphere forming efficiency). Mammosphere assays wereperformed in triplicate and repeated three times independently.

Metabolic Flux Analysis: Extracellular acidification rates and oxygenconsumption rates were analyzed using the Seahorse XFe96 analyzer(Agilent/Seahorse Bioscience, USA). Cells were maintained in DMEMsupplemented with 10% FBS (fetal bovine serum), 2 mM GlutaMAX, and 1%Pen- Strep. Twenty-thousand breast cancer cells were seeded per well,into XFe96-well cell culture plates, and incubated at 37° C. in a 5% CO2humidified atmosphere for at least 12 hours to allow cell attachment.After about 24 hours, MCF7 cells were washed in pre-warmed XF assaymedia, or for OCR measurement, XF assay media supplemented with 10 mMglucose, 1 mM Pyruvate, 2 mM L-glutamine, and adjusted at 7.4 pH. Cellswere then maintained in 175 μL/well of XF assay media at 37° C., in anon-CO2 incubator for 1 hour. During the incubation time, 25 μL of 80 mMglucose, 9 μM oligomycin, and 1M 2-deoxyglucose (for ECAR measurement)or 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (forOCR measurement), was loaded in XF assay media into the injection portsin the XFe96 sensor cartridge. Measurements were normalized by proteincontent (SRB assay) and Hoechst 33342 content. Data sets were analyzedusing XFe96 software and GraphPad Prism software, using one-way ANOVAand Student's t-test calculations. All experiments were performed inquintuplicate, three times independently.

Cell Cycle Analysis by FACS: Cell-cycle analysis was performed on theATP-high and ATP-low cell sub-populations, by FACS analysis using theAttune NxT Flow Cytometer. Briefly, after trypsinization, there-suspended cells were incubated with 10 ng/ml of Hoescht solution(Thermo Fisher Scientific) for 40 min at 37° C. under dark conditions.Following a 40 minute period, the cells were washed and re-suspended inPBS Ca/Mg for acquisition or in sorting buffer [1× PBS containing 3%(v/v) FBS and 2 mM EDTA] for FACS. 50,000 events were analyzed percondition. Gated cells were manually-categorized into cell-cycle stages.

Statistical Significance: All analyses were performed with GraphPadPrism 6. Data were represented as mean±SD (or ±SEM where indicated). Allexperiments were conducted at least 3 times independently, with >3technical replicates for each experimental condition tested (unlessstated otherwise, e.g., when representative data is shown).Statistically significant differences were determined using theStudent's t-test or the analysis of variance (ANOVA) test. For thecomparison among multiple groups, one-way ANOVA was used to determinestatistical significance. p<0.05 was considered significant and allstatistical tests were two-sided: p* <0.05; p** <0.01; p*** <0.005;p**** <0.0001.

Bioinformatic analysis: Unbiased label-free proteomics, comparing2D-monolayers and 3D-mammospheres, was carried out as previouslydescribed, using MCF7 and T47D breast cancer cell lines. Informaticsanalysis was performed using a variety of publicly available of GEODataSets (GSE36953; GSE2034; GSE59000; GSE55470), archived in the NCBIdatabase, related to 3D growth, metastasis and circulating tumor cells(CTCs). Gene expression profiling data was extracted from these GEODataSets. HeatMaps were generated with QIAGEN OmicSoft Suite Software.Volcano plots were produced by examining the annotations present inOncoLand Metastatic Cancer (QIAGEN OmicSoft Suite). In addition,functional “core analyses” was performed using Ingenuity PathwayAnalysis Software (IPA; QIAGEN), on annotated genes. Gene co-expressionprofiles were extracted from The Metastatic Breast Cancer ProjectProvisional (2020), using cBioPortal (https://www.cbioportal.org/); mRNAexpression profiling (RNA Seq V2 RSEM) was carried via RNA-sequencing ofmetastatic breast cancer samples from 146 patients.

Kaplan-Meier (K-M) analysis: To perform K-M analysis on ATP5F1C, we usedan open-access online survival analysis tool to interrogatepublicly-available microarray data from up to 3,951 breast cancerpatients. For this purpose, we primarily analyzed data from ER(+)patients. Biased array data were excluded from the analysis. Thisallowed us to identify ATP5F1C (also known as ATP5C1), as a significantprognostic marker. Hazard-ratios were calculated, at the bestauto-selected cut-off, and p-values were calculated using the Log-ranktest and plotted in R. K-M curves were generated online using theK-M-plotter (as high-resolution TIFF files), using univariate analysis:

https ://kmplot.com/analysis/index.php?p=service&cancer=breast.

This approach allowed for directly performing in silico validation ofATP5F1C as a marker of tumor recurrence (RFS, replapse-free survival)and distant metastasis (DMFS, distant metastasis-free survival). Thelatest 2020 version of the database was utilized for all these analyses.

Cell Migration Assays: Briefly, 2.5×104 cells in 0.5 ml of serum-freeDMEM with 0.1% BSA were added to the wells of 8-μm pore, non-coatedmembrane modified Boyden chambers (Transwells). The lower chamberscontained 10% fetal bovine serum in DMEM to serve as a chemo-attractant.Cells were incubated at 37° C. and allowed to migrate throughout thecourse of 6 h. Noninvasive cells were removed from the upper surface ofthe membrane by scrubbing with cotton swabs. Chambers were stained in0.5% crystal violet diluted in 100% methanol for 30-60 min, rinsed inwater and examined under a bright-field microscope. Values for invasionand migration were obtained by counting five fields per membrane (20xobjective) and represent the average of three independent experiments.Note that Transwells, pre-coated with extracellular matrix (namelyMatrigel), were used to measure aggressive cell invasion and preventsimple cell migration.

Metastasis Assays: The chick embryo metastasis assay was performed byINOVOTION (Societe: 811310127), La Tronche-France. According to theFrench legislation, no ethical approval is needed for scientificexperimentations using oviparous embryos (decree n° 2013-118, Feb. 1,2013; art. R-214-88). Animal studies were performed under animalexperimentation permit N° 381029 and B3851610001 to INOVOTION.Fertilized White Leghorn eggs were incubated at 37.5° C. with 50%relative humidity for 9 days. Greater than 20 eggs were processed foreach experimental condition. At that moment (E9), the chorioallantoicmembrane (CAM) was dropped down by drilling a small hole through theeggshell into the air sac, and a 1 cm² window was cut in the eggshellabove the CAM. The MDA-MB-231 tumor cell line was cultivated in DMEMmedium supplemented with 10% FBS and 1% penicillin/streptomycin. On dayE9, cells were detached with trypsin, washed with complete medium andsuspended in graft medium. After ATP-based cell sorting byflow-cytometry, an inoculum of 30,000 cells was added onto the CAM ofeach egg (E9) and then eggs were randomized into groups. On day E18, a 1cm² portion of the lower CAM was collected to evaluate the number ofmetastatic cells in 8 samples per group (n=10). Genomic DNA wasextracted from the CAM (commercial kit) and analyzed by qPCR withspecific primers for Human Alu sequences. Calculation of Cq for eachsample, mean Cq and relative amounts of metastases for each group aredirectly managed by the Bio-Rad® CFX Maestro software. Non-injected eggswere also evaluated in parallel, as a negative control for specificity.A one-way ANOVA analysis with post-tests was performed on all the data.

The terminology used in the description of embodiments of the presentapproach is for the purpose of describing particular embodiments onlyand is not intended to be limiting. As used in the description and theappended claims, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. The present approach encompasses numerous alternatives,modifications, and equivalents as will become apparent fromconsideration of the following detailed description.

It will be understood that although the terms “first,” “second,”“third,” “a),” “b),” and “c),” etc. may be used herein to describevarious elements of the present approach, and the claims should not belimited by these terms. These terms are only used to distinguish oneelement of the present approach from another. Thus, a first elementdiscussed below could be termed an element aspect, and similarly, athird without departing from the teachings of the present approach.Thus, the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc.are not intended to necessarily convey a sequence or other hierarchy tothe associated elements but are used for identification purposes only.The sequence of operations (or steps) is not limited to the orderpresented in the claims.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art. It will be further understood that terms,such as those defined in commonly used dictionaries, should beinterpreted as having a meaning that is consistent with their meaning inthe context of the present application and relevant art and should notbe interpreted in an idealized or overly formal sense unless expresslyso defined herein. All publications, patent applications, patents andother references mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification is controlling.

Also, as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the present approach described herein can beused in any combination. Moreover, the present approach alsocontemplates that in some embodiments, any feature or combination offeatures described with respect to demonstrative embodiments can beexcluded or omitted.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claim. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable value,such as, for example, an amount or concentration and the like, is meantto encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount. A range provided herein for a measurable value mayinclude any other range and/or individual value therein.

Having thus described certain embodiments of the present approach, it isto be understood that the scope of the appended claims is not to belimited by particular details set forth in the above description as manyapparent variations thereof are possible without departing from thespirit or scope thereof as hereinafter claimed.

What is claimed is:
 1. A purified composition of hyper-proliferativecancer stem cells comprising a sub-population of cells in a human cancercell population, the cancer cell population expressing a range offluorescent signals in response to a fluorescent adenosine triphosphate(ATP) imaging probe, and the sub-population of cells expressing an upperportion of the range of ATP-based fluorescent signals.
 2. Thecomposition of claim 1, wherein the upper portion comprises the top 10%of ATP-based fluorescent signals.
 3. The composition of claim 1, whereinthe upper portion comprises the top 5% of ATP-based fluorescent signals.4. The composition of claim 1, wherein the composition is positive forone of a CD44 marker and an ALDH marker.
 5. The composition of claim 1,wherein the composition comprises circulating tumor cells (CTC).
 6. Thecomposition of claim 1, wherein the composition is frozen.
 7. A purifiedcell composition comprising a cancer stem cell sub-population stainedwith a fluorescent adenosine triphosphate (ATP) imaging probe andexpressing a target portion of an ATP-based fluorescent signal range ofa cancer cell population.
 8. The composition of claim 7, wherein thecancer cell population expresses a range of ATP-based fluorescentsignals, and the target portion of the ATP-based fluorescent signalrange is one of an upper portion of the ATP-based fluorescent signalsand a lower portion of the ATP-based fluorescent signals.
 9. Thecomposition of claim 8, wherein the target portion is one of the top 10%of ATP-based fluorescent signals and the top 5% of ATP-based fluorescentsignals.
 10. The composition of claim 8, wherein the target portion isone of the bottom 10% of ATP-based fluorescent signals and the bottom 5%of ATP-based fluorescent signals.
 11. The composition of claim 9,wherein the composition is positive for one of a CD44 marker and an ALDHmarker.
 12. The composition of claim 1, wherein the sub-population ofcells is stained with a fluorescent ATP imaging dye.
 13. A purifiedcomposition of cells obtained by staining a human cancer cell populationwith a fluorescent adenosine triphosphate (ATP) imaging probe,separating a fraction of the human cancer cell population having atarget portion of ATP-based fluorescent signals, and purifying theseparated cells.
 14. The composition of claim 13, wherein the targetportion comprises one of the top 10% of ATP-based fluorescent signals,the top 5% of ATP-based fluorescent signals, the bottom 10% of ATP-basedfluorescent signals, and the bottom 5% of ATP-based fluorescent signals.15. The composition of claim 13, wherein the target portion comprisesone of the top 10% of ATP-based fluorescent signals, the top 5% ofATP-based fluorescent signals, and the separated cells are positive forone of a CD44 marker and an ALDH marker.
 16. The composition of claim13, wherein the fluorescent imaging probe comprises ATP-Red-1.
 17. Amethod of ATP-based cell fractionation, the method comprising: stainingcells in a cell population with a fluorescent adenosine triphosphate(ATP) imaging probe that fluoresces when bound to ATP; measuring theATP-based fluorescent signals of the stained cells in the cellpopulation; and separating the stained cells based on a target portionof ATP-based fluorescent signals.
 18. The method of claim 17, whereinthe target portion comprises one of the top 10% of ATP-based fluorescentsignals, the top 5% of ATP-based fluorescent signals, the bottom 10% ofATP-based fluorescent signals, and the bottom 5% of ATP-basedfluorescent signals.
 19. The method of claim 17, wherein separating thestained cells based on target portion of ATP-based fluorescent signalscomprises fluorescence-activated cell sorting (FACS) gating of thetarget portion of ATP-based fluorescent signals.
 20. The method of claim19, wherein the gates are set to collect at least one of (i) the stainedcells having the top 10% of measured ATP-based fluorescent signals, and(ii) the stained cells having the bottom 10% of measured ATP-basedfluorescent signals.
 21. The method of claim 17, wherein the fluorescentATP imaging probe comprises ATP-Red
 1. 22. The method of claim 17,wherein the cell population is derived from one of blood, urine, saliva,tumor tissue, non-cancerous tissue, and a metastatic lesion.
 23. Themethod of claim 17, further comprising at least one of measuring ALDHactivity of separated cells, measuring anchorage-independent growth ofseparated cells, measuring the mitochondrial mass of separated cells,measuring the glycolytic and oxidative mitochondrial metabolism ofseparated cells, measuring the cell cycle progression and proliferativerate of separated cells, and measuring the poly-ploidy of separatedcells.
 24. A method for separating and collecting metabolicallyhyper-proliferative cells from a cell population, the method comprising:staining cells in a cell population with an ATP-labeling dye, whereinthe ATP-labeling dye fluoresces when bound to ATP; measuring theATP-based fluorescent signals of the stained cells in the cellpopulation; separating the stained cells based on the measured ATP-basedfluorescent signals; and collecting at least a portion of the separatedcells having a measured ATP-based fluorescent signal one of above apredetermined threshold and below a predetermined threshold.
 25. Theseparating and collecting method of claim 24, wherein the ATP-labelingdye comprises ATP-Red
 1. 26. The separating and collecting method ofclaim 24, wherein the predetermined threshold comprises a percentage ofan upper portion of the measured ATP-based fluorescent signals.
 27. Theseparating and collecting method of claim 26, wherein the predeterminedthreshold comprises one of the top 25%, the top 20%, the top 15%, thetop 10%, the top 5%, the top 2%, and the top 1%.
 28. The separating andcollecting method of claim 24, wherein separating and collecting isperformed using fluorescence-activated cell sorting (FACS).
 29. Theseparating and collecting method of claim 24, wherein the separatedcells are further separated based on a second marker.
 30. The separatingand collecting method of claim 29, wherein the second marker comprisesone of CD44(+), CD133(+), ESA(+), ALDEFLOUR(+), MitoTracker-High,EpCAM(+), CD90(+), CD34(+), CD29(+), CD73(+), CD90(+), CD105(+),CD106(+), CD166(+), and Stro-1(+).
 31. The separating and collectingmethod of claim 30, wherein separating cells based on a second markeroccurs at least one of (i) prior to staining cells in the cellpopulation with the ATP-labeling dye, and (ii) after staining cells inthe cell population with the ATP-labeling dye.
 32. The separating andcollecting method of claim 31, wherein the second marker comprises anantibody coated on magnetic beads.
 33. The separating and collectingmethod of claim 24, further comprising staining the cells in the cellpopulation with a second marker, and wherein the measuring the ATP-basedfluorescent signals of the stained cells in the cell population occursafter staining with the second marker and the ATP-labeling dye.
 34. Amethod for identifying and treating cancer stem cells in a biologicsample, the method comprising: obtaining a biologic sample from apatient; staining cells in the biologic sample with an ATP-labeling dye,wherein the ATP-labeling dye fluoresces when bound to ATP; measuring theATP-based fluorescent signals of the stained cells in the cellpopulation; comparing the measured ATP-based fluorescent signals to apredetermined threshold indicating the presence of cancer stem cells;and if the measured ATP-based fluorescent signals exceeds thepredetermined threshold, administering to the patient at least oneATP-depletion therapeutic.
 35. The method of claim 34, wherein theATP-depletion therapeutic comprises one of Doxycycline, Tigecycline,Azithromycin, Pyrvinium pamoate, Atovaquone, Bedaquiline, Niclosamide,Irinotecan, Actinonin, CAPE, Berberine, Brutieridin, Melitidin,Oligomycin, AR-C155858, a Mitoriboscin, a Mitoketoscin, a Mitoflavoscin,a TPP-derivative, dodecyl-TPP, 2-Butene-1,4-bis-TPP, Doxycyclineconjugated with a fatty acid, and a combination of Doxycycline,Azithromycin and Ascorbic acid.
 36. A method of testing a candidatecompound for anti-cancer activity, the method comprising: staining acancer cell population with an ATP-labeling dye, wherein theATP-labeling dye fluoresces when bound to ATP; measuring the ATP-basedfluorescent signals of the stained cells; separating the stained cellsbased on a target portion of ATP-based fluorescent signals to prepare ahyper-active cancer cell sub-population; administering the candidatecompound to the hyper-active cancer cell sub-population; and measuringthe effect of the candidate compound on the hyper-active cancer cellsub-population.
 37. The method of claim 36, wherein the ATP-labeling dyecomprises ATP-Red
 1. 38. The method of claim 36, wherein the targetportion of ATP-based fluorescent signals comprises one of the top 25%,the top 20%, the top 15%, the top 10%, the top 5%, the top 2%, and thetop 1%.
 39. The method of claim 36, wherein the hyper-active cancer cellsub-population is positive for one of a CD44 marker an ALDH marker. 40.The method of claim 36, further comprising at least one of measuringALDH activity of the hyper-active cancer cell sub-population, measuringanchorage-independent growth of the hyper-active cancer cellsub-population cells, measuring the mitochondrial mass of thehyper-active cancer cell sub-population, measuring the glycolytic andoxidative mitochondrial metabolism of the hyper-active cancer cellsub-population, measuring the cell cycle progression and proliferativerate of the hyper-active cancer cell sub-population, and measuring thepoly-ploidy of the hyper-active cancer cell sub-population.
 41. A methodof diagnosing and preventing a risk of metastasis in a cancer patient,comprising: determining the expression levels of ABCA2, ATP5F1C, COX20,NDUFA2, and UQCRB, in a biologic sample of the patient's cancer;comparing the detected expression levels to baseline expression levelsof ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB, in a non-cancerous biologicsample from the patient; and if the detected expression levels exceedthe baseline expression levels, administering an ATP-depletion compoundto the patient.
 42. The method of claim 41, wherein the ATP-depletioncompound comprises one of Doxycycline, Tigecycline, Azithromycin,Pyrvinium pamoate, Atovaquone, Bedaquiline, Niclosamide, Irinotecan,Actinonin, CAPE, Berberine, Brutieridin, Melitidin, Oligomycin,AR-C155858, a Mitoriboscin, a Mitoketoscin, a Mitoflavoscin, aTPP-derivative, dodecyl-TPP, 2-Butene-1,4-bis-TPP, Doxycyclineconjugated with a fatty acid, and a combination of Doxycycline,Azithromycin and Ascorbic acid.
 43. A kit for identifying circulatingtumor cells (CTCs) in a biologic sample, the kit comprising reagents foridentifying an up-regulation of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRBin the biologic sample.
 44. The kit of claim 43, wherein the reagentscomprise at least one antibody directed at one of ABCA2, ATP5F1C, COX20,NDUFA2, and UQCRB.
 45. A method for detecting circulating tumor cells(CTCs) in a biologic sample, the method comprising: determining theexpression levels of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB, in thebiologic sample; and indicating the presence of CTCs if the determinedexpression levels are upregulated relative to a control.
 46. The methodof claim 45, wherein the biologic sample comprises one of blood, urine,saliva, tumor tissue, non-cancerous tissue, and a metastatic lesion. 47.The method of claim 45, further comprising separating CTCs from thebiologic sample by staining the sample with a fluorescent ATP-labelingdye, measuring the ATP-based fluorescent signals of the stained sample;separating the stained sample based on a target portion of ATP-basedfluorescent signals; and collecting the cell sub-population having thetarget portion of AATP-based fluorescent signals.
 48. The method ofclaim 47, wherein the target portion of ATP-based fluorescent signalscomprises one of the top 25%, the top 20%, the top 15%, the top 10%, thetop 5%, the top 2%, and the top 1%.